Molecular basis of AKAP79 regulation by calmodulin

Patel, Neha; Stengel, Florian; Aebersold, Ruedi; Gold, Matthew G.

doi:10.1038/s41467-017-01715-w

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Published: 22 November 2017

Molecular basis of AKAP79 regulation by calmodulin

Nature Communications volume 8, Article number: 1681 (2017) Cite this article

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Abstract

AKAP79/150 is essential for coordinating second messenger-responsive enzymes in processes including synaptic long-term depression. Ca²⁺ directly regulates AKAP79 through its effector calmodulin (CaM), but the molecular basis of this regulation was previously unknown. Here, we report that CaM recognizes a ‘1-4-7-8’ pattern of hydrophobic amino acids starting at Trp79 in AKAP79. Cross-linking coupled to mass spectrometry assisted mapping of the interaction site. Removal of the CaM-binding sequence in AKAP79 prevents formation of a Ca²⁺-sensitive interface between AKAP79 and calcineurin, and increases resting cellular PKA phosphorylation. We determined a crystal structure of CaM bound to a peptide encompassing its binding site in AKAP79. CaM adopts a highly compact conformation in which its open Ca²⁺-activated C-lobe and closed N-lobe cooperate to recognize a mixed α/3₁₀ helix in AKAP79. The structure guided a bioinformatic screen to identify potential sites in other proteins that may employ similar motifs for interaction with CaM.

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Introduction

Calmodulin (CaM) is a Ca²⁺-sensing protein that is expressed in all eukaryotic cells. It mediates many essential processes driven by Ca²⁺, including long-term changes in synaptic connections in the brain¹, apoptosis², and immune responses³. CaM is a bilobal protein, with each lobe consisting of two EF hands that adopt an open conformation upon binding Ca²⁺. Ca²⁺/CaM binds and modulates a wide range of proteins. For example, Ca²⁺/CaM-binding relieves autoinhibition in the phosphatase calcineurin and in Ca²⁺/CaM-dependent protein kinase II (CaMKII). CaM possesses a highly flexible structure that is capable of recognizing a diversity of interaction motifs. Many recognition motifs for Ca²⁺/CaM fall within two major classes⁴. Proteins including myosin light chain kinase⁵ and calcineurin present amphiphilic helices with anchor hydrophobic residues at positions 1, 8, and 14, whereas proteins including CaMKII⁶ bind CaM through shorter helical sequences with the hydrophobic anchors at position 1, 5, and 10. High-resolution crystal and NMR structures have established how both lobes of CaM act in concert to coordinate either 1-5-10 or 1-8-14 class motifs^7,8. Outside of these predominant classes, in a few cases Ca²⁺/CaM binds in an extended conformation to longer recognition sequences⁹. For example, Munc13 presents a 1-5-8-26 pattern of hydrophobic amino acids for interaction with Ca²⁺/CaM¹⁰. Prior to this study, in all cases where CaM binding depends on Ca²⁺, all four EF hands have been found to bind Ca²⁺ in high-resolution structures. In addition, with a single exception¹¹, the terminal hydrophobic anchor positions are separated by no fewer than eight amino acids. Despite decades of progress in understanding CaM recognition sequences, CaM-binding sites in some Ca²⁺/CaM-regulated proteins have eluded mapping. One such protein is AKAP79, which was the focus of this investigation.

AKAP79 (rodent ortholog AKAP150, gene name AKAP5) is a prototypic A-kinase-anchoring protein (AKAP) that fulfills key physiological roles, including supporting long-term synaptic depression¹², directing calcineurin for NFAT dephosphorylation¹³, and controling release and response to insulin¹⁴. AKAP79 engages in multiple protein–protein interactions, including with cAMP-dependent protein kinase (PKA)¹⁵, calcineurin¹⁶ and Ca²⁺/CaM¹⁷. The calcineurin-binding site on AKAP79 has been proposed as a drug target^14,18 for treating diabetes^14,18. It also interacts with the membrane bilayer through a combination of N-terminal polybasic regions¹⁹ and double palmitoylation^20,21. Constitutive binding sites for calcineurin and PKA have been mapped within AKAP79: PKA binds to an amphipathic helix near to the anchoring protein’s C-terminus¹⁵, whereas calcineurin binds to the sequence PIAIIITD between AKAP79 residues 337 and 343¹⁹. Initial mapping of these two anchoring sites has been corroborated by high-resolution structures^22,23,24, leading to functional investigations using AKAP79 variants containing targeted ablations^12,14. Binding of Ca²⁺/CaM within the first 153 amino acids of AKAP79²⁵ antagonizes interactions between the tandem basic regions of the protein and acidic headgroups of the membrane bilayer¹⁹. Ca²⁺/CaM also stabilizes interactions between AKAP79 and calcineurin by triggering a secondary interface with the phosphatase that involves elements in the N-terminus of AKAP79²⁵, although it is not clear how CaM triggers this interface. AKAP79 contains no sequences conforming to 1-5-10 or 1-8-14 motifs, and the absence of a precise location for CaM binding has impeded progress in understanding how CaM regulates AKAP79.

We set out to determine the CaM-binding site in AKAP79 by exploiting the rapidly developing technique cross-linking coupled to mass spectrometry (XL-MS). The general approach is to cross-link proteins under native conditions, followed by detection and exact sequence identification of cross-linked peptides by MS (Fig. 1a)²⁶. The interlinking pattern between CaM and AKAP79 served as a foundation for identifying and characterizing the CaM-binding site in AKAP79.

Results

Analysis of cross-linking CaM in complex with AKAP79

We first purified AKAP79 bound to the dimerization and docking (D/D) domain of type IIα PKA regulatory subunits (Supplementary Fig. 1a, b). This complex, ‘AKAP79-D/D’, was mixed with purified CaM in an equimolar ratio and incubated with the homobifunctional amine-reactive cross-linker disuccinimidyl suberate (DSS) in the presence of 100 μM Ca²⁺. Coomassie staining (Fig. 1b) and immunoblotting against AKAP79 (Fig. 1c) or CaM (Fig. 1d) after electrophoresis on denaturing gels confirmed that cross-linking covalently coupled CaM and AKAP79. The most prominent band at 80 kD corresponds to cross-linked AKAP79-CaM heterodimers, whereas the band at 160 kD represents stabilization of 2AKAP79-2CaM tetramers, which is consistent with native MS and gel filtration studies, indicating that AKAP79 dimerizes^25,27. To determine which lysines had cross-linked within the complex, the sample was digested with trypsin, and cross-linked peptides were identified using liquid chromatography (LC)-MS/MS. A total of 213 peptides were identified from three biological replicates, each shot in technical duplicates. These consist of 47 interlinks between CaM and AKAP79, 148 links between AKAP79 peptides (intralinks or links between homodimeric AKAP proteins), 13 CaM-CaM intralinks, and 5 links between symmetrical peptides, indicating interlinks between AKAP79 homodimers (Supplementary Table 1). Such symmetrical dimeric interlinks were detected at three positions in AKAP79 (83-83, 50-50, 48-48), which is consistent with previous reports that AKAP79 dimerizes in a parallel conformation involving N-terminal contacts²⁵. An exemplar MS/MS sequencing spectrum is shown for a peptide linked between K94_CaM and K99_AKAP79 (Fig. 1e). The overall pattern of intralinks (grey) and interlinks (black) within and between AKAP79 and CaM is illustrated in Fig. 1f.

Interlinks between AKAP79 and CaM were restricted to lysines between residues 41 and 177 in the N-terminus of AKAP79 (Fig. 1g)²⁵. To narrow down the location of the CaM-binding site on AKAP79, we considered the frequency with which particular lysine pairs were observed in cross-linked peptides. Multiple detections of a particular link may arise as enzymatic digestion may lead to peptides of varying lengths containing the same cross-linking sites. In addition, the data set contains the data pooled from biological replicates (potential additional multiple identifications of cross-linked peptides originating from different charge states or technical duplicates were counted as the same unique cross-linked peptide). A high frequency of a particular lysine pairing therefore strongly indicates that the two amino acids are likely to be optimally positioned for cross-linking under native conditions. In this case, 66% of all interlinks were mediated between K94 in CaM (K94_CaM) and either K90_AKAP79 (4 detections, Fig. 1g), K96_AKAP79 (7 detections, Fig. 1g) or K99_AKAP79 (16 detections, Fig. 1g). The identification of this cross-linking hot-spot provided us with a reference point to triangulate the position of the CaM-binding site in AKAP79.

Location of the CaM-binding site in AKAP79

The cross-linking data suggested that CaM binds in the vicinity of AKAP79 lysines 90, 96, and 99. AKAP79 contains a predicted helical region between amino acids 79–86 in the vicinity of this cross-linking hot spot. To examine whether this could correspond to the CaM-binding site in AKAP79, we used a complementary method - the amplified luminescent proximity homogenous assay (alpha)-screen technique-to scan the N-terminus of AKAP79 for sequences that could mediate interactions with CaM. Incubation of GST-AKAP79 (1–153), but not control GST, with biotinylated CaM brings streptavidin donor and anti-GST acceptor beads within 200 Å, which can be detected by measuring emission between 520 and 620 nm following illumination at 680 nm (Fig. 2a, b). Ca²⁺triggered CaM association with GST-AKAP79 (1-153) but not with GST alone (lanes 2 and 4, Fig. 2b, p = 2.2 × 10⁻⁸). We screened peptides derived from the N-terminus of AKAP79 for their ability to reduce alphascreen emission. Twenty 20-mer peptides were synthesized corresponding to a walk in increments of 5 amino acids along the N-terminus of AKAP79 beginning at 21–40 (peptide ‘A’, Fig. 2c) and ending at 116–135 (peptide ‘T’, Fig. 2c). The effect of each peptide at 100 nM concentration on CaM–AKAP79 (1–153) interaction is shown in Fig. 2d as log2 emission ratios (with/without peptide). Consistent with the cross-linking data, the only two peptides encompassing residues 79 and 86 most effectively inhibited the interaction. Peptide M (positions 71–90) reduced the signal to 0.13 ± 0.02 compared to untreated samples; and peptide N (76–95) reduced the signal to 0.21 ± 0.03 of the untreated emission. No other peptides reduced emission below 0.60 relative to untreated samples. There is one other predicted helical sequence in the AKAP79 N-terminus, which falls between residues 33 and 48, but no peptides spanning this region produced a marked reduction in alphascreen signal (peptides C-F, Fig. 2d).

To verify that CaM binds to the helix spanning residues 79–86, we first performed pull-down experiments using lysates from human embryonic kidney 293T (HEK293T) cells expressing full-length FLAG-tagged AKAP79 variants (Fig. 3a). Wild-type AKAP79 associates with both CaM sepharose, and cAMP agarose by virtue of its ability to interact with type II regulatory (RII) subunits of PKA (lane 1, Fig. 3a). Control experiments confirmed that CaM sepharose pull-down of WT AKAP79 was Ca²⁺-dependent (Supplementary Fig. 2b). Deletion of the AKAP79 anchoring helix (Δ391-400) prevents its association with cAMP agarose but does not affect binding to CaM sepharose (lane 4, Fig. 3a), as expected. We tested two novel deletion mutants of AKAP79 lacking one or other of the two predicted helical sequences. Removal of residues 33–48 did not affect either CaM sepharose or cAMP agarose binding (lane 2, Fig. 3a). However, deletion of residues 79–86 abolished binding to CaM sepharose but not cAMP agarose binding (lane 3, Fig. 3a), which is consistent with the results of cross-linking (Fig. 1g) and peptide scanning (Fig. 2d). Removal of residues 79–86 also abolished interaction between GST-AKAP79 (1–153) and biotinylated CaM, according to the alphascreen assay (Supplementary Fig. 2c).

The predicted helical sequence spanning AKAP79 residues 79–86 is shown in Fig. 3b. The helix contains hydrophobic amino acids in a 1-4-7-8 pattern (yellow, Fig. 3b) that does not conform to conventional 1-5-10 and 1-8-14 interaction motifs. Several basic amino acids lie at the immediate C-terminus of this hydrophobic cluster, and these are also highly conserved across the AKAP5 gene family (sequence LOGO, Fig. 3b). To determine whether these basic amino acids contribute to the interaction, we compared how effectively peptides of different lengths could disrupt CaM–AKAP79 interaction. We calculated inhibition constants (K _i–the concentration for half-maximal inhibition) for each peptide after incubating at different concentrations with GST-AKAP79 (1–153)/biotin-CaM and measuring alphascreen emission. The peptides analyzed are shown at the bottom of Fig. 3b. Whereas a 9-mer peptide spanning positions 1–9 exhibited low efficacy with a K _i = 5.1 ± 1.2 μM (black line, Fig. 3c), addition of the conserved amino acids G77 and A78 at the N-terminus brought the peptide efficacy into the nanomolar range (K _i = 170 ± 40 nM, red line, Fig. 3c). Extension to a 16-mer by inclusion of the polybasic sequence ‘RRKRS’ increased potency further with K _i = 75 ± 8 nM (black line, Fig. 3d), suggesting that these basic amino acids are involved in the interaction. Extension to 20-mer (red line, Fig. 3d, K _i = 32 ± 2 nM) led to a similar fold increase in potency, but additional extension to the 26-mer produced little further improvement (black line, Fig. 3e, K _i = 22 ± 1).

Some CaM targets, such as Munc13, present a major helical hydrophobic sequence with a shorter hydrophobic motif some distance downstream—at position 26 relative to the first anchor position¹⁰ in the case of Munc13. The primary sequence of AKAP79 does not reveal any strong candidates for a second hydrophobic motif but we nevertheless tested whether mutating the best candidate, L101, affects the potency of the 26-mer peptide. L101A substitution led to no significant reduction in potency (K _i = 24 ± 2, red line, Fig. 3e). Overall the cross-linking, pull-down and alphascreen experiments show that AKAP79 contains an unconventional 1-4-7-8 motif beginning at W79 that is principally responsible for Ca²⁺-dependent CaM binding, supported by a stretch of ~10 basic-rich amino acids following the motif.

CaM-dependence of interaction between calcineurin and AKAP79

Having established the location of the CaM-binding site in AKAP79, we next explored how the CaM-binding sites in AKAP79 and calcineurin contribute to Ca²⁺/CaM-dependent interaction between the N-terminus of AKAP79 and calcineurin. CaM could conceivably drive these interactions by binding either or both of its interaction sites in calcineurin and AKAP79. We first engineered two point mutations into calcineurin (I396A/I400A) that CaM-calcineurin peptide crystal structures²⁸ indicate would render the phosphatase deficient in binding CaM (Fig. 4a). Assays of phosphatase activity against a phosphorylated peptide confirmed that the I396A/I400A calcineurin variant is activated with a half-maximal CaM concentration (K_CaM) >20-fold higher (k = 670 ± 60 nM, red in Fig. 4b) than the WT phosphatase (K _CaM = 30 ± 12 nM, black in Fig. 4b). Further details of curve fitting to determine these values are provided in Supplementary fig. 3. Next, we performed calcineurin pull-downs using GST-fusions to the N-terminus (1–153) of AKAP79 (Fig. 4c) in the presence of Ca²⁺ and 100 nM CaM. Removing the 1-4-7-8 motif in AKAP79 (Δ79-86) led to marked reductions in pull down of either WT (lanes 2 & 3, Fig. 4c) or I396A/I400A calcineurin (lanes 5 & 6, Fig. 4c). Densitometry indicated a ~10-fold reduction in anti-calcineurin immunoblot signal upon removal of the 1-4-7-8 motif (Fig. 4c, n = 3, p = 0.0097). Conversely, phosphatase pull-down using GST-WT AKAP79 (1–153) was hardly altered when WT calcineurin was substituted with the I396A/I400A variant (lanes 2 & 5, Fig. 4c). These experiments suggest that the CaM-binding site in AKAP79 is required for driving the Ca²⁺-dependent calcineurin-AKAP79 interface, whereas calcineurin activation is not required. It is important to note that these experiments do not rule out the possibility that CaM binding to the phosphatase is required since this occurs at markedly lower concentrations than are required to activate the phosphatase²⁹. We also examined the effect of removing the 1-4-7-8 motif in live HEK293T cells using A-Kinase Activity Reporter 4 (AKAR4)³⁰. The FRET efficiency of AKAR4 increases upon phosphorylation of a central phosphorylation site such that the emission ratio at 535/485 nm presents a real-time readout of phosphorylation at a PKA site (Fig. 4d). We found that the baseline FRET ratio was increased by 8.1 ± 1.4 %—indicative of higher resting PKA phosphorylation—when the cells were co-transfected with Δ79–86 AKAP79 rather than WT AKAP79 (Fig. 4e, n = 12, p = 8 × 10⁻⁵). In sum, the experiments outlined in Fig. 4 show that removing the CaM-binding site in AKAP79 has consequences for PKA phosphorylation of cellular substrates.

Structure of CaM bound to its AKAP79 recognition site

To understand how the atypical ‘1-4-7-8’ motif in AKAP79 is able to bind CaM, we attempted to co-crystallize CaM with peptides spanning the motif. Co-crystallization was achieved with a peptide corresponding to AKAP79 amino acids 77–92. X-ray diffraction images were collected to a resolution of 1.7 Å enabling determination of the crystal structure by molecular replacement using the C-lobe of CaM in complex with MARCKS peptide¹¹. The structure was autobuilt using Buccaneer³¹, and refined in Coot³² and PHENIX³³. The assymetric unit contains two copies of CaM–AKAP79 peptide complex (Supplementary Fig. 4a). For each copy, AKAP79 amino acids 78–88 are visible in the electron density (Supplementary Fig. 4b). The two copies of the complex are similar with overall root mean standard deviation (rmsd) for Cα atoms = 0.834 Å. They show closer similarity between their CaM C-lobes (amino acids 80–145, rmsd = 0.276 Å), and in the AKAP79 peptide (0.597 Å), than between N-lobes (amino acids 2–79, rmsd = 0.87 Å). Unexpectedly, while all of the C-lobe EF hands coordinate Ca²⁺ ions in the open conformation (light blue, Fig. 5a, b), the N-lobe EF hands are in a closed metal ion-free conformation (dark blue, Fig. 5a, b ). Consistent with the absence of Ca²⁺ ions in the N-lobes, CaM amino acids 56–59 (part of the second N-lobe EF-hand) are not visible in the electron density.

Although only the C-lobes coordinate Ca²⁺, remarkably both CaM lobes engage the AKAP79 peptide (orange, Fig. 5b). All four hydrophobic side-chains of the AKAP79 1-4-7-8 motif are accommodated within a hydrophobic pocket formed by the coming together of both CaM lobes (Fig. 5b). Trp79 in AKAP79 (W79_AKAP79) is deeply buried in a cleft within the CaM C-lobe, which is the canonical binding position for the first hydrophobic anchor amino acid. To determine the relative importance of different amino acids within the 1-4-7-8 motif for CaM association, we compared the ability of mutant peptides derived from AKAP79 77–96 to disrupt interactions between biotin-CaM and GST-AKAP79 (1–153). Whereas wild-type peptide reduced emission to 0.03 ± 0.01 relative to untreated samples, substitution of Trp79 with alanine greatly reduced the potency of the peptide (p = 0.0013) with a small reduction in emission to 0.78 ± 0.06 relative to the untreated condition (Fig. 5c). The substitution V86A slightly reduced potency (0.17 ± 0.04 remaining emission, p = 0.019, compared to the WT peptide), while the helix-destabilizing mutation L82P also markedly reduced the potency of the peptide (0.61 ± 0.08 relative emission, p = 0.0034, Fig. 5c). Alanine substitutions at positions ‘4’ (L82) and ‘7’ (L85) had no significant effect on potency, indicating that they are less essential for the interaction (lanes 3 and 4, Fig. 5c). We also translated these findings into the context of full-length AKAP79 on the basis that studies with peptides might not capture the full complexity of the interaction. CaM sepharose pull-down experiments with purified full-length AKAP79-RIIα D/D complexes support a key role for W79_AKAP79 in mediating association with CaM. Whereas wild-type AKAP79 efficiently bound to CaM sepharose (Fig. 5d), both the deletion of positions 79–86, or substitution of tryptophan 79 for alanine, abolished binding (Fig. 5d).

The binding mode is unusual following anchoring of Trp79_AKAP79 within the CaM C-lobe. Strikingly, the AKAP79 polypeptide twists away from CaM after position 86 (orange, Fig. 5b), whereas most CaM-interacting proteins present single straight α-helices for interaction. CaM adopts a highly compact conformation to accommodate AKAP79. We searched for structural homologs of full-length CaM taken from the complex using the DALI server and returned no structural matches spanning both lobes with rmsd < 3 Å (Supplementary Table 2). This analysis confirms that, in this crystal structure, CaM adopts a conformation that has not been observed before.

The binding helix contains 3₁₀ helical character

The Ramachandran plot for amino acids in AKAP79 shows that six amino acids in the structure exhibit the geometry of right-handed helices (positions 80–85, Fig. 5e). Whereas positions 80–83 exhibit α-helical backbone geometry (diamonds, Fig. 5e), positions 84 and 85 possess more positive ψ angles that are characteristic of 3₁₀ helices (black triangles, Fig. 5e). Analysis of the hydrogen-bonding pattern within AKAP79 confirms that the AKAP79 helix exhibits 3₁₀ helicity at its C-terminus. The L85_AKAP79 backbone amino group H-bonds with the carbonyl group of L82_AKAP79, and the equivalent atom in V86_AKAP79 also H-bonds with the carbonyl group of the amino acid three residues upstream (Fig. 5f, striped lines). The characteristic triangular 3₁₀ helical conformation between amino acids 83 and 86 is most clearly visible when looking down the barrel of the AKAP79 helix (Fig. 5g). The backbone groups of pairs 79–83 and 80–84 engage in α-helical H-bonds (Fig. 5f, dotted lines). This overall helical geometry enables AKAP79 to present an amphipathic helix, in which all four hydrophobic positions, including W79_AKAP79 and V86_AKAP79 (Fig. 5f) are projected from the same side of the helix. This is the first example of a CaM-binding helix containing 3₁₀ helical character.

Analysis of Ca²⁺-dependence of the CaM–AKAP79 interaction

Although there is no close structural homolog of the conformation of full-length CaM observed in complex with AKAP79, both the N- and C-lobes separately resemble conformations observed in other crystal structures of the Ca²⁺ sensor (Supplementary Table 2). The C-lobe in particular has more than 20 structural homologs within an rmsd of 1 Å, including the C-lobe of CaM in complex with the 1-8-14 motif of calcineurin²⁸ (rmsd 0.5 Å, Cα positions 80–147). However, in cases where the C-lobe adopts a similar conformation, the N-lobe is always markedly different with both EF hands coordinating Ca²⁺. For example, Fig. 6a shows differences in both the conformation and position of the N-lobes of CaM in complex with AKAP79 (blue) or calcineurin (black) when the CaM C-lobes are aligned. Nevertheless, this structural analysis indicates that the C-lobe, in complex with AKAP79, adopts an essentially canonical open Ca²⁺-bound conformation that is able to accommodate a linear pattern of hydrophobic side-chains. In this case, the four hydrophobic side-chains of the amphipathic 1-4-7-8 motif engage in van der Waals contacts with this binding pocket. W79_AKAP79 is sandwiched between M124_CaM and M144_CaM; L82_AKAP79 packs against F92_CaM; L85_AKAP79 engages the side-chains of both F12_CaM and L39_CaM; and V86_AKAP79 contacts both M145_CaM and I85_CaM (Fig. 6b). The side-chains of A78_AKAP79 and T87_AKAP79 also contact hydrophobic patches on the surface of the C-lobe (Fig. 6b), whereas the side-chains of S81_AKAP79 and T87_AKAP79 form direct H-bonds with this same lobe (dotted lines, Fig. 6b).

The isolated N-lobe possesses fewer close structural homologs. The highest scoring match is to the N-lobe of CaM in complex with an IQ motif from myosin VI (rmsd 1 Å, Supplementary Table 2)–the class of motif that CaM binds in the Ca²⁺-free state³⁴. All of the CaM N-lobe structural homologs retrieved by DALI, including structures of apo CaM³⁵, are closed and Ca²⁺-free (Supplementary Table 2). Although CaM also employs a closed N-lobe to engage myosin VI, the relative orientations of the N- and C-lobes are markedly different in this complex (black, Fig. 6c) compared to with AKAP79 (blue, Fig. 6c). For example, E7_CaM, Q8_CaM and E11_CaM, which engage in H-bonds with AKAP79, are not involved in the interaction with myosin VI and instead face into solution in the CaM-myosin complex. Closer inspection of the CaM–AKAP79 peptide complex reveals that N-lobe H-bonds, involving residues including E7_CaM, Q8_CaM and E11_CaM, enable CaM to accommodate the atypical AKAP79 helix (Fig. 6d). Notably, the side-chain of E11_CaM H-bonds to the backbone carbonyl of S81_AKAP79, thus satisfying an H-bond lost in the transition from α to 3₁₀ helix (marked by an asterisk in Fig. 5f). Similarly, the side-chain of Q8_CaM fulfills a helix-capping function in H-bonding to the backbone carbonyl of L85_AKAP79 (Fig. 6d). In addition to this network of H-bonds, N-lobe residues L39_CaM and F12_CaM form a hydrophobic pocket that enables van der Waals interactions with the side-chain of L85_AKAP79 (Supplementary Fig. 4c).

The crystal structure suggests that Ca²⁺ binding to the CaM N-lobe is not required for binding AKAP79. To test this, we compared the ability of CaM mutants unable to bind Ca²⁺ in either the N-lobe (CaM12) or C-lobe (CaM34) to inhibit interaction between biotin-CaM and GST-AKAP79 (1–153). CaM12 inhibited the interaction with a similar K _i (91 ± 6 nM, red line, Fig. 6e) to wild-type CaM (108 ± 8 nM, black line), consistent with the notion that Ca²⁺ binding to the CaM N-lobe is not required for interaction with AKAP79. CaM34 (K _i = 930 ± 130 nM, blue line, Fig. 6e) was a significantly less potent inhibitor of the interaction, which supports the idea that generation of the open conformation of the C-lobe by Ca²⁺ binding enables interaction with AKAP79. Although Ca²⁺ binding to the CaM N-lobe is apparently not required for association with AKAP79, we found that CaM still associates with AKAP79 at relatively low Ca²⁺ concentrations: alphascreen measurements show that the half-maximal concentrations of free Ca²⁺ [Ca₅₀] for CaM binding to AKAP79 and calcineurin are similar (Fig. 6f). Biotin-CaM bound to GST-calcineurin with Ca_50 =0.41 ± 0.01 μM (green, Fig. 6f), whereas the concentration was slightly increased to 0.64 ± 0.01 μM when the same experiment was performed with GST-AKAP79 (1–153) (orange, Fig. 6f). Ca²⁺ binding to CaM was highly cooperative in both cases (calcineurin, n = 3.2 ± 0.2; AKAP79, n = 4.4 ± 0.3). The small difference in Ca²⁺ sensitivity suggests that at certain intermediate cellular Ca²⁺ concentrations, calcineurin may be active while in complex with AKAP79, while the CaM-binding site in the anchoring protein remains unoccupied. Overall, the structural and alphascreen data show despite an unusual binding mode, Ca²⁺ sensitivity of the interaction with AKAP79 is similar to interactions with canonical CaM target sequences.

CaM adopts a highly compact conformation to bind AKAP79

There are striking differences between the binding mode of CaM with AKAP79 in comparison to that observed in complexes containing class 1-5-10 and class 1-8-14 sequences. In complex with AKAP79, CaM adopts a highly compact conformation (Fig. 7a) exposing only 8700 Å³ surface area. This reflects the unusually short spacing⁹ of 11.9 Å between Cα atoms of the terminal hydrophobic anchor positions W79_AKAP79 and V86_AKAP79. In comparison, CaM presents 9300 Å³ total surface area when binding to the 1-5-10 motif of CaMKII (Fig. 7b, 14.4 Å between anchor positions), and 9900 Å³ when binding the 1-8-14 motif of calcineurin (Fig. 7c, 19.5 Å between anchor positions). Another marked difference between AKAP79 and 1-5-10/1-8-14 motifs is the path of the recognition motif through CaM. The binding sequence in AKAP79 does not form a single straight helix, but twists out from CaM after L85_AKAP79 with dihedral angles characteristic of a β-sheet for positions equivalent to ‘9’ and ’10’. Concomitantly, CaM buries only 740 Å³ in the interaction with AKAP79 (Fig. 7a). When the three binding modes are viewed from the same angle looking down on the amphiphilic side of the binding helix, only the class 1-5-10 and 1-8-14 motifs pass underneath the CaM linker (grey, Fig. 7a–c). As a result, the interfaces with CaMKII (1400 Å³, Fig. 7b) and calcineurin (1200 Å³, Fig. 7c) are much larger.

Discussion

This investigation has generated multiple findings concerning the structure and function of AKAP79 regulation by CaM, and the results have wider implications for regulation by CaM. The CaM site in AKAP79 had eluded discovery for 20 years following the initial observation that AKAP79 binds CaM¹⁷. Mapping the site has enabled us to perform the first experiments using CaM-binding-deficient variants of AKAP79 to establish that Ca²⁺/CaM-dependent interactions between AKAP79 and calcineurin require the CaM-binding site in AKAP79. We also demonstrated that ablating the CaM-binding site in AKAP79 increases resting PKA phosphorylation in HEK293T cells. The binding mode observed between CaM and AKAP79 further expands the documented binding capabilities of CaM⁹. The CaM–AKAP79 crystal structure reveals three abilities of CaM: it can adopt an extremely compact conformation (8700 Å surface area, Fig. 7a); associate with sequences containing 3₁₀ helical turns; and bind a target using both lobes where only the C-lobe is in the open Ca²⁺-activated conformation (Fig. 5a). Overall, the study highlights the remarkable flexibility of CaM, and the utility of XL-MS for identifying protein—protein interaction sites.

Binding of Ca²⁺/CaM to AKAP79 can have two effects: it can trigger an additional interface with calcineurin (Fig. 4c), and it has previously been shown to reduce binding of the anchoring protein to vesicles containing acidic phospholipids¹⁹. The second polybasic sequence in AKAP79 (region ‘B’ between positions 75–108) is particularly important for PIP₂ binding¹⁹. Six basic amino acids fall within or at the immediate C-terminus of the 1-4-7-8 motif (lysines 83 and 90; arginines 84, 88, 89 and 91) within region B. It appears likely that CaM occludes these six basic amino acids upon binding, thereby releasing the anchoring protein from acidic phospholipid headgroups. The Ca²⁺/CaM-dependent calcineurin interaction site also falls within the N-terminus of AKAP79. In this study, experiments with the reporter AKAR4 indicate that removing the 1-4-7-8 motif within AKAP79 elevates resting PKA phosphorylation in HEK293T cells (Fig. 4e). However, much remains to be understood concerning the purpose of the Ca²⁺/CaM-sensitive AKAP79-calcineurin interface. Comparison of knockin mice containing targeted ablations of the constitutive PKA and calcineurin anchoring sites in AKAP150 has proved to be highly valuable for disentangling the roles of these sites in insulin signaling and long-term depression^12,14. A similar approach incorporating mice containing knockin variants of AKAP150 lacking the 1-4-7-8 motif may now be applied now the site is mapped.

We were curious to investigate whether the binding motif in AKAP79 is a one-off, or if it might represent the first example of a hitherto unappreciated class of CaM interaction motif (Fig. 7d). On the basis of the AKAP79-CaM crystal structure, we developed a position-specific scoring matrix to search the human proteome for potential 1-4-7-8 motifs with Scansite 3. The best 100 matching sequences are listed in Supplementary Table 3. The list is likely to contain many false positives but contains several sites that warrant further investigation. The list includes five potential 1-4-7-8 motifs in other AKAPs: three similar sequences within Gravin, one in BIG1, and one in BIG2. A previous study identified a ~200 amino acid region of Gravin that can bind CaM without precisely defining the binding sites³⁶: all three potential 1-4-7-8 motifs (with initial tryptophan anchors at positions 610, 759, and 804) fall within this region. The search also retrieved a potential 1-4-7-8 motif present in seven of the ten voltage-dependent Ca²⁺ channel pore-forming α subunits (Ca_V1 and Ca_v2-type subunits, Supplementary Table 3). The sequence falls within a long helix in a short intracellular loop between transmembrane helices S4 and S5 in domain II³⁷ that moves upon voltage-dependent activation³⁸. Conceivably access to this site could be altered upon voltage-dependent activation of the channels, providing a mechanism for coupling Ca²⁺ and depolarization to channel regulation. Additional putative 1-4-7-8 sites of note include one potential site close to the membrane-tethered C-terminus of BCL-2, a key anti-apoptotic protein³⁹. It will be exciting to see which of this long list of putative sites prove to be high affinity CaM-binding sites with physiological significance.

Methods

Protein expression and purification

All protein purification steps were performed at 4 °C. To express AKAP79 in bacteria in a non-aggregated form, we cloned the D/D domain (residues 1–45) of human PKA RIIα into pGEX6P1 for expression as a PreScission-cleavable fusion at the N-terminus of GST, and included a second cistron containing N-terminally 6xHis-tagged AKAP79. Primer sequences are listed in Supplementary table 4. Co-expression of AKAP79-D/D was induced in BL21 (DE3) E. coli (Novagen) at OD₆₀₀ = 0.5 with 0.4 mM IPTG, and the cells were harvested after 3 h at 37 °C. The cell pellet was resuspended in lysis buffer (30 mM Tris-HCl pH 8, 300 mM NaCl, 10 mM imidazole, 1 mM benzamidine, one EDTA-free protease inhibitor tablet/100 mL (Roche), 0.1 mg mL⁻¹ lysozyme), sonicated briefly, and clarified by centrifugation at 40,000 × g for 30 min. The supernatant was collected and incubated with Ni-NTA agarose (Qiagen) for 2 h prior to elution in 30 mM Tris-HCl pH 7.5, 300 mM NaCl, 300 mM imidazole, 1 mM benzamidine. The eluted protein was buffer exchanged into glutathione sepharose binding buffer (25 mM Tris pH 7.4, 500 mM NaCl, 2 mM DTT, 1 mM EDTA, 1 mM Benzamidine) using a Sephadex G-25 column, before 3 h incubation with glutathione sepharose 4B (GE Life Sciences). AKAP79-D/D was eluted by overnight incubation with PreScission protease (GE Life Sciences). In the final purification step, AKAP79-D/D complex eluted between 0.4 and 0.5 column volumes from a Superdex-200 gel filtration column in 20 mM Hepes pH 7.5 and 200 mM NaCl (Supplementary Fig. 1b). AKAP79- Δ79–86 and W79A variants, in complex with the RIIα D/D domain, were expressed and purified in the same way. Gel filtration elution profiles confirm that deletion of amino acids 79–86 does not affect the peak elution volume of AKAP79- RIIα D/D upon gel filtration (Supplementary Fig. 1b). GST fusion proteins (Supplementary Fig. 1d, e, f), and calcineurin (WT and I396A/I400A; Supplementary Fig. 1h, i) were purified, using affinity to glutathione sepharose and size exclusion chromatography (SEC)²⁵, after expression in BL21 (DE3) E. coli.

For crystallography, human CaM was cloned into pET28-a for expression of untagged protein in E. coli BL21 (DE3) cells. CaM was expressed by overnight incubation at 37 °C in 3 L auto-inducing media⁴⁰, yielding a cell pellet of ~30 g. The pellet was resuspended in 150 mL lysis buffer (25 mM Tris pH 7.4, 500 mM NaCl, 1 mM EDTA, 2 mM DTT, 1 mM Benzamidine, 0.1 mg/mL⁻¹ lysozyme), and sonicated briefly prior to high-speed centrifugation (20,000 × g, 30 min). The clarified lysate was supplemented up to 5 mM CaCl₂, before incubation with phenyl sepharose (GE Life Sciences) for 3 h. After washing (50 mM Tris pH 7.4, 1 mM CaCl₂, 500 mM NaCl), CaM was eluted in 10 mL EDTA buffer (50 mM Tris pH 7.4, 1 mM EDTA). The eluate was diluted in buffer containing 20 mM Tris pH 8.5, 2 mM DTT, 1 mM EDTA to a final NaCl concentration of ~20 mM, enabling binding to a 1 mL Resource Q column (GE Life Sciences). CaM was eluted from the Resource Q column with a salt/pH gradient by varying the proportions of a mixture of buffers A (20 mM Tris pH 8.5, 20 mM NaCl, 1 mM benzamidine, 2 mM DTT, 1 mM EDTA) and B (20 mM Tris pH 7.2, 1 M NaCl, 1 mM benzamidine, 2 mM DTT, 1 mM EDTA). Finally, CaM was dialysed into water and lyophilized using a vacuum concentrator. This protocol yields ~1.5 mg purified CaM per L culture (Supplementary Fig. 1c). Vectors for expressing CaM12 (D20A/D56A) and CaM34 (D93A/D129A) variants were generated using site-directed mutagenesis of pET28-a-CaM, and these variants were expressed in the same way and purified using phenyl sepharose and gel filtration. For XL-MS experiments, CaM was expressed with a PreScission-cleavable GST tag, and purified by affinity to glutathione sepharose, PreScission cleavage and SEC²⁵. Coomassie stained SDS-PAGE gels show the purity of biotinylated CaM, CaM, CaM12, and CaM34 (Supplementary Fig. 1g, j).

Cross-linking coupled to mass spectrometry

AKAP79-D/D and CaM were mixed at a 1:1 molar ratio to a final concentration of 1 mg/mL in 100 μL buffer containing 20 mM Hepes pH 7.5, 200 mM NaCl and 100 μM CaCl₂. Cross-linking was inititated by adding 2 μL H12/D12 DSS (Creative Molecules) from a 25 mM stock prepared in DMSO. After 30 min incubation at 30 °C in a thermomixer set to 300 rpm, reactions were terminated by addition of 5 μL from a 1 M stock of ammonium bicarbonate. Each 100 μg cross-linked sample was enzymatically digested with trypsin and subsequently enriched by SEC for cross-linked peptides. LC-MS/MS analysis was carried out on an Orbitrap Elite mass spectrometer (Thermo Scientific). The data were searched using xQuest⁴¹ in ion-tag mode with a precursor mass tolerance of 10 p.p.m. For matching of fragment ions, tolerances of 0.2 Da for common-ions and 0.3 Da for cross-link ions were applied. Fragment ions were analyzed in an ion trap, trading lower mass accuracy for increased sensitivity and measuring speed. Potential cross-links were statistically validated from fragment ion spectra and false discovery rates (FDR) were assigned using xProphet ⁴². Cross-linked AKAP79-D/D-CaM samples were prepared in biological triplicate, and each of these was measured as technical duplicates. For each experiment, the final data set was restricted to high-confidence unique cross-links with both ID scores >20 and FDRs <0.05. Spectra of potential crosslinks were also visually inspected to ensure good matches of ion series on both cross-linked peptide chains for the most abundant peaks.

Pull-down assays

For pull-down assays involving N-terminally FLAG-tagged AKAP79 variants, HEK293T cells (American Type Culture Collection) were initially cultured in DMEM supplemented with GlutaMAX, Penicillin/Streptomycin, and 10% (v/v) fetal bovine serum (all supplied by Gibco). 10 cm diameter plates were transfected at 60 % confluence using 4 μg pcDNA3.1-FLAG-AKAP79 and 16 μL Fugene 6 (Promega). Cells were transfected with WT, Δ33–48, Δ79–86, or Δ391–400 (‘ΔPKA’) AKAP79. 3 days after transfection, cells were collected and lysed in 1 mL HEK293T buffer (30 mM Tris pH 7.4, 150 mM NaCl, 1% Igepal CA-630, 1 mM EDTA, 1 mM benzamidine, 2 mM DTT). Following sonication, lysates were clarified by centrifugation, then split equally for incubation with either 20 μL CaM sepharose 4B (GE Healthcare) or 20 μL 8-AEA-cAMP-agarose (Biolog) beads. After overnight incubation, beads were washed with 6 × 1 mL HEK293T buffer, and finally protein was eluted with 40 μL 1 × LDS sample buffer (ThermoFisher). For CaM sepharose pull-downs, the clarified lysate and wash buffer were supplemented with 3 mM CaCl₂. FLAG-AKAP79 variants were detected using rabbit anti-FLAG primary antibody (Sigma), and goat anti-rabbit-HRP-conjugate secondary antibody (Bio-Rad). An additional experiment was performed to confirm that binding of WT AKAP79 to CaM-sepharose is Ca²⁺-dependent. In this case, HEK293T cells were transfected with pcDNA3.1-AKAP79-V5 (WT or Δ79–86). The lysate from each plate was clarified by centrifugation, divided into two, and one half of the material was supplemented with 3 mM CaCl₂. Each half was incubated with 20 μL CaM sepharose 4B overnight, then washed with 6 × 1 mL HEK293T buffer (supplemented with 3 mM CaCl₂, where appropriate) the following morning, and eluted in 1 × LDS sample buffer. AKAP79-V5 was detected using anti-V5-HRP-conjugate antibody (ThermoFisher). For pull-down experiments using AKAP79-D/D complexes purified from bacteria, pull-down was performed using a similar procedure. In this case, 0.5 μg purified protein was added in each pull-down. Bound protein was detecting by first eluting with Ca²⁺-free HEK293T buffer containing 1 mM EDTA, and then immunoblotting with anti-AKAP79 (Millipore) antibody.

To determine which CaM-binding site enables Ca²⁺-dependent interaction between the N-terminus of AKAP79 and calcineurin, 1 μg GST-AKAP79 1–153 (WT or Δ79-86) was incubated with 2 μg calcineurin (WT or I396A/I400A) in pull-down buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% Igepal CA-630, 0.25% sodium deoxycholate, 1 mM EDTA, 1 mM benzamidine) containing 100 nM CaM. The samples were supplemented with 3 mM CaCl₂ where appropriate. The samples were incubated with 2 μL magnetic glutathione bead slurry (Pierce) for 4 h, washed 4 times in pull-down buffer supplemented with 50 nM CaM, and 3 mM CaCl₂ where appropriate. Finally, proteins were eluted from the magnetic beads in 50 μL 1 × LDS sample buffer. Calcineurin pull-down was detected using mouse anti-calcineurin antibody (Catalog number 610259, BD Biosciences, 1:500 dilution).

Amplified luminescent proximity homogeneous assay screens

To prepare biotin-labeled CaM for use in alphascreen assays, 1 mg purified CaM was incubated with a two-fold molar excess of EZ link NHS Biotin (Pierce) in 20 mM Na Hepes pH 7.5, 200 mM NaCl. Labeling was terminated after 30 min at 22 °C by supplementing with Tris-HCl pH 7.4 to a final concentration of 50 mM. Biotininylated CaM was separated from free biotin by SEC yielding >200 μL biotininylated CaM at 80 μM. In general, assays were performed in 50 μL buffer consisting of 20 mM HEPES pH 7.2, 100 mM NaCl, 0.1% BSA, 1 mM EGTA, 5 mM MgCl₂, 1 mM CaCl₂. CaCl₂ was excluded where appropriate. A PEPscreen was ordered from Sigma containing the following peptides (~2 mg each): 20-mer peptides walking through the N-terminus of AKAP79 in 5-amino-acid increments, starting at 21–40 and ending at 116–135 for a total of 20 peptides; and 20-mer peptides corresponding to AKAP79 (77–96) containing either WT sequence or the substitutions W79A, L82A, L85A, V86A, L82P. The following high (>95%) purity peptides were ordered from Biomatik: ‘9-mer’ (AKAP79 positions 79–87), ’11-mer’ (77–87), ’16-mer’ (77–92), ’20-mer’ (77–96), and ’26-mer’ (77–102) both with and without the substitution L101A.

For all alphascreen experiments, GST-AKAP79 (1–153) and biotinylated CaM were first preincubated for 1 h at 22 °C in the presence of the appropriate peptide of unbiotinylated CaM additives. Biotin-CaM was included at 10 nM in all mixtures, whereas GST-AKAP79 (1–153) varied between experiments as follows: 3 nM (Fig. 2d, Fig. 3c–e, Supplementary Fig. 2c), 6 nM (Fig. 5f), and 8 nM (Fig. 2d). To determine the K _Ca for CaM binding to calcineurin and AKAP79, the CaCl₂ concentration was varied to produce different free Ca²⁺ concentrations on the basis of calculations using maxchelator⁴³. After initial incubations, anti-GST acceptor beads (PerkinElmer) were added at 10 ng/μL. After 30 min, streptavidin donor beads were also added at 10 ng/μL. After 1 h further incubation with vigorous shaking in a thermomixer (1200 r.p.m.), light emission at 568 nm was measured following excitation with a 680 nm laser using a Mithras LB 940 (Berthold Technologies).

Protein crystallization

Peptide corresponding to AKAP79 positions 77–92 (GAWASLKRLVTRRKRS) was synthesized at 96% purity (Biomatik). Lyophilized CaM was resuspended in 10 mM CaCl₂ at 7.5 mg/mL and mixed with a 3:1 molar excess of peptide. Crystals were grown at 22 °C using hanging-drop vapor diffusion by mixing 1 μL of CaM solution with 1 μL mother liquor (2.4 M ammonium sulphate, 50 mM citrate pH 5.4, 0.3 M NDSB-195). After growth for one month, crystals were flash-frozen in liquid N₂ using mother liquor supplemented with 3.5 M ammonium sulphate.

X-ray diffraction data collection and processing

The diffraction data were collected at the Diamond Light Source (Harwell, UK) beamline I24. Diffraction images were processed using iMosflm⁴⁴ and merging/scaling of reflection intensities was performed by AIMLESS⁴⁵. AIMLESS indicated an appropriate resolution limit of 1.7 Å (1.73–1.7 Å shell: I/σI = 3.1, CC_1/2 = 0.552). Initial phases were obtained by molecular replacement in PHASER⁴⁶ using the C-lobe of CaM (residues 85–148, PDB 1IWQ) as the search model. This confirmed that two copies of the CaM–AKAP79 peptide complex are present in the asymmetric unit. Automated model building was performed in Buccaneer³¹. The model was refined by rounds of manual model building in Coot³² and automated refinement in PHENIX³³. Refinement was performed using 20 translation/libration/screw groups defined automatically by PHENIX. The structure was validated with MolProbity⁴⁷. The data collection and refinement statistics are shown in Table 1. Stereo images of the 2F_o−F_c electron density map clipped to the AKAP79 peptide are shown in Supplementary Fig. 4b.

Table 1 X-ray diffraction data collection and refinement statistics

Full size table

Structure and sequence analysis

Structural representations of the complex (chains B and D of the asymmetric unit unless otherwise stated) were rendered using Pymol (www.pymol.org). Secondary structure prediction was performed using Jpred3⁴⁸. Alignments, and LOGO plots of AKAP5 sequences were retrieved from the ensembl database (www.ensembl.org). Distances were calculated using NCONT, and solvent accessibility was determined using AREAIMOL⁴⁹. Automated H-bond identification was performed using UCSF Chimera⁵⁰, 3D structural homologs were identified using the DALI server⁵¹, and pairwise structural alignments were performed using GESAMT⁴⁹.

A-kinase activity reporter assays

Fluorescence-based recordings of PKA activity were performed in HEK293T cells attached to 96-well plates. HEK293T cells were cultured and transfected in the same way as for pull-down assays. In this case, cells were transfected at 60 % confluence in 6-well plates using 1 μg DNA/4 μL Fugene 6 per well. The DNA consisted of a mixture of 0.3 μg pcDNA3.1-FLAG-AKAP79 (WT or Δ79-86) and 0.7 μg AKAR4 DNA. 2 days after transfection, the cells were detached using trypsin and resuspended in 3 mL supplemented DMEM, then re-plated at 100 μL/well in 96-well black-walled plates (Molecular Probes) that had been coated overnight with poly-l-lysine (Sigma). The following morning, the media was exchanged for PBS (100 μL per well), and FRET ratios were immediately determined using a Mithras LB 940 plate-reader (Berthold Technologies) controlled by a computer running Mikrowin 2000 software. To enable recordings using AKAR4³⁰, the plate reader was fitted with a 420 nm excitation filter (Berthold no. 39452), and emission filters at 485 nm (no. 40271) and 535 nm (no. 40273).

Protein phosphatase assays

Protein phosphatase activity was determined by measuring phosphate release from phosphorylated RII peptide (DLDVPIPGRFDRRVpSVAAE) synthesized at >95% purity (Biomatik). Assays were performed with either WT or I396A/I400A calcineurin at 20 nM in 50 μL reactions containing different concentrations of CaM in phosphatase assay buffer (20 mM Na Hepes, 100 mM KCl, 0.1% BSA, 1 mM EGTA, 6 mM MgCl₂, 1 mM CaCl₂). Each assay was initiated by adding pRII substrate (75 μM final concentration), and terminated after 1 h at 30 °C by addition of 50 μL Biomol Green (Enzo Life Sciences). Phosphate release was determined by measuring absorbance at 630 nm using an ELx800 spectrophotometer (Biotek) after color had been allowed to develop for 30 min at 22 °C.

Motif-based scanning for 1-4-7-8 CaM-binding sites

Scansite 3 was used to perform searches for putative 1-4-7-8 class CaM-binding motifs⁵². To identify potential 1-4-7-8 motifs, we assembled a position-specific scoring matrix file guided by contacts between AKAP79 and CaM in the crystal structure: the strongest weighting was assigned to the first anchor tryptophan, followed by hydrophobic amino acids at positions 4, 7, and 8. Conservative substitutions that could be accommodated according to the crystal structure were assigned intermediate weightings. The basic amino acids lysine and arginine were equally weighted at positions ‘5’ and ‘6’. The full matrix is shown in supplementary Fig. 5. The matrix was used to search Homo sapiens protein sequences within the SwissProt database (20252 total input proteins). 570 predicted sites were found, with a median score = 0.633, and median absolute deviation = 0.108. The top 100 highest scoring predicted sites are listed in supplementary Table 3.

Statistical analysis

Densitometry was performed using ImageJ⁵³. Two-tailed student’s t tests were used to calculate p values for the differences between mean values (n ≥ 3 for all experiments). Densitometry was performed using ImageJ⁵³. Curve fitting to determine K _Ca and K _i values was performed by fitting data to Hill functions by iterative least squares minimization using ORIGIN software (OriginLab). K _CaM co-efficients for calcineurin activation were calculated by fitting to Michaelis-Menten functions containing a cooperativity variable (Supplementary Fig. 3).

Data availability

The coordinate and structure factor data of the CaM—AKAP79 (77–92) complex has been deposited in the Protein Data Bank (PDB) with the accession code 5NIN. Uncropped images of blots and gels are shown in Supplementary Fig. 6. The other data are available from the corresponding author upon reasonable request.

References

Lisman, J., Yasuda, R. & Raghavachari, S. Mechanisms of CaMKII action in long-term potentiation. Nat. Rev. Neurosci. 13, 169–182 (2012).
CAS PubMed PubMed Central Google Scholar
Orrenius, S., Zhivotovsky, B. & Nicotera, P. Regulation of cell death: the calcium-apoptosis link. Nat. Rev. Mol. Cell. Biol. 4, 552–565 (2003).
Article CAS PubMed Google Scholar
Crabtree, G. R. Calcium, calcineurin, and the control of transcription. J. Biol. Chem. 276, 2313–2316 (2001).
Article CAS PubMed Google Scholar
Rhoads, A. R. & Friedberg, F. Sequence motifs for calmodulin recognition. FASEB J. 11, 331–340 (1997).
CAS PubMed Google Scholar
Blumenthal, D. K. et al. Identification of the calmodulin-binding domain of skeletal muscle myosin light chain kinase. Proc. Natl Acad. Sci. USA 82, 3187–3191 (1985).
Article ADS CAS PubMed PubMed Central Google Scholar
Hanley, R. M. et al. Functional analysis of a complementary DNA for the 50-kilodalton subunit of calmodulin kinase II. Science 237, 293–297 (1987).
Article ADS CAS PubMed Google Scholar
Ikura, M. et al. Solution structure of a calmodulin-target peptide complex by multidimensional NMR. Science 256, 632–638 (1992).
Article ADS CAS PubMed Google Scholar
Meador, W. E., Means, A. R. & Quiocho, F. A. Modulation of calmodulin plasticity in molecular recognition on the basis of x-ray structures. Science 262, 1718–1721 (1993).
Article ADS CAS PubMed Google Scholar
Tidow, H. & Nissen, P. Structural diversity of calmodulin binding to its target sites. FEBS J. 280, 5551–5565 (2013).
Article CAS PubMed Google Scholar
Rodriguez-Castaneda, F. et al. Modular architecture of Munc13/calmodulin complexes: dual regulation by Ca2+ and possible function in short-term synaptic plasticity. EMBO J. 29, 680–691 (2010).
Article CAS PubMed Google Scholar
Yamauchi, E., Nakatsu, T., Matsubara, M., Kato, H. & Taniguchi, H. Crystal structure of a MARCKS peptide containing the calmodulin-binding domain in complex with Ca2+ -calmodulin. Nat. Struct. Biol. 10, 226–231 (2003).
Article CAS PubMed Google Scholar
Sanderson, J. L., Gorski, J. A. & Dell’Acqua, M. L. NMDA receptor-dependent LTD requires transient synaptic incorporation of Ca(2)(+)-permeable AMPARs mediated by AKAP150-anchored PKA and calcineurin. Neuron 89, 1000–1015 (2016).
Article CAS PubMed PubMed Central Google Scholar
Murphy, J. G. et al. AKAP-anchored PKA maintains neuronal L-type calcium channel activity and NFAT transcriptional signaling. Cell Rep. 7, 1577–1588 (2014).
Article CAS PubMed PubMed Central Google Scholar
Hinke, S. A. et al. Anchored phosphatases modulate glucose homeostasis. EMBO J. 31, 3991–4004 (2012).
Article CAS PubMed PubMed Central Google Scholar
Carr, D. W., Stofko-Hahn, R. E., Fraser, I. D., Cone, R. D. & Scott, J. D. Localization of the cAMP-dependent protein kinase to the postsynaptic densities by A-kinase anchoring proteins. Characterization of AKAP 79. J. Biol. Chem. 267, 16816–16823 (1992).
CAS PubMed Google Scholar
Coghlan, V. M. et al. Association of protein kinase A and protein phosphatase 2B with a common anchoring protein. Science 267, 108–111 (1995).
Article ADS CAS PubMed Google Scholar
Faux, M. C. & Scott, J. D. Regulation of the AKAP79-protein kinase C interaction by Ca2+ /Calmodulin. J. Biol. Chem. 272, 17038–17044 (1997).
Article CAS PubMed Google Scholar
Gold, M. G., Gonen, T. & Scott, J. D. Local cAMP signaling in disease at a glance. J. Cell. Sci. 126, 4537–4543 (2013).
Article CAS PubMed PubMed Central Google Scholar
Dell’Acqua, M. L., Faux, M. C., Thorburn, J., Thorburn, A. & Scott, J. D. Membrane-targeting sequences on AKAP79 bind phosphatidylinositol-4, 5-bisphosphate. EMBO J. 17, 2246–2260 (1998).
Article PubMed PubMed Central Google Scholar
Delint-Ramirez, I., Willoughby, D., Hammond, G. R., Ayling, L. J. & Cooper, D. M. Palmitoylation targets AKAP79 protein to lipid rafts and promotes its regulation of calcium-sensitive adenylyl cyclase type 8. J. Biol. Chem. 290, 13041 (2015).
Article CAS PubMed PubMed Central Google Scholar
Woolfrey, K. M., Sanderson, J. L. & Dell’Acqua, M. L. The palmitoyl acyltransferase DHHC2 regulates recycling endosome exocytosis and synaptic potentiation through palmitoylation of AKAP79/150. J. Neurosci. 35, 442–456 (2015).
Article PubMed PubMed Central CAS Google Scholar
Gold, M. G. et al. Molecular basis of AKAP specificity for PKA regulatory subunits. Mol. Cell. 24, 383–395 (2006).
Article CAS PubMed Google Scholar
Kinderman, F. S. et al. A dynamic mechanism for AKAP binding to RII isoforms of cAMP-dependent protein kinase. Mol. Cell. 24, 397–408 (2006).
Article CAS PubMed PubMed Central Google Scholar
Li, H. et al. Balanced interactions of calcineurin with AKAP79 regulate Ca2+ -calcineurin-NFAT signaling. Nat. Struct. Mol. Biol. 19, 337–345 (2012).
Article PubMed PubMed Central CAS Google Scholar
Gold, M. G. et al. Architecture and dynamics of an A-kinase anchoring protein 79 (AKAP79) signaling complex. Proc. Natl Acad. Sci. USA 108, 6426–6431 (2011).
Article ADS CAS PubMed PubMed Central Google Scholar
Leitner, A., Faini, M., Stengel, F. & Aebersold, R. Crosslinking and mass spectrometry: An integrated technology to understand the structure and function of molecular machines. Trends Biochem. Sci. 41, 20–32 (2016).
Article CAS PubMed Google Scholar
Gao, S., Wang, H. Y. & Malbon, C. C. AKAP12 and AKAP5 form higher-order hetero-oligomers. J. Mol. Signal. 6, 8 (2011).
Article CAS PubMed PubMed Central Google Scholar
Dunlap, T. B. et al. Stoichiometry of the calcineurin regulatory domain-calmodulin complex. Biochemistry 53, 5779–5790 (2014).
Article CAS PubMed Google Scholar
Quintana, A. R., Wang, D., Forbes, J. E. & Waxham, M. N. Kinetics of calmodulin binding to calcineurin. Biochem. Biophys. Res. Commun. 334, 674–680 (2005).
Article CAS PubMed Google Scholar
Depry, C., Allen, M. D. & Zhang, J. Visualization of PKA activity in plasma membrane microdomains. Mol. Biosyst. 7, 52–58 (2011).
Article CAS PubMed Google Scholar
Cowtan, K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. D Biol. Crystallogr. 62, 1002–1011 (2006).
Article PubMed CAS Google Scholar
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).
Article CAS PubMed PubMed Central Google Scholar
Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr. 68, 352–367 (2012).
Article CAS PubMed PubMed Central Google Scholar
Xie, X. et al. Structure of the regulatory domain of scallop myosin at 2.8 A resolution. Nature 368, 306–312 (1994).
Article ADS CAS PubMed Google Scholar
Kuboniwa, H. et al. Solution structure of calcium-free calmodulin. Nat. Struct. Biol. 2, 768–776 (1995).
Article CAS PubMed Google Scholar
Tao, J., Shumay, E., McLaughlin, S., Wang, H. Y. & Malbon, C. C. Regulation of AKAP-membrane interactions by calcium. J. Biol. Chem. 281, 23932–23944 (2006).
Article CAS PubMed Google Scholar
Wu, J. et al. Structure of the voltage-gated calcium channel Cav1.1 at 3.6 A resolution. Nature 537, 191–196 (2016).
Article ADS CAS PubMed Google Scholar
Catterall, W. A. Voltage-gated calcium channels. Cold Spring Harb. Perspect. Biol. 3, a003947 (2011).
Article PubMed PubMed Central CAS Google Scholar
Bonneau, B., Prudent, J., Popgeorgiev, N. & Gillet, G. Non-apoptotic roles of Bcl-2 family: the calcium connection. Biochim. Biophys. Acta. 1833, 1755–1765 (2013).
Article CAS PubMed Google Scholar
Studier, F. W. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234 (2005).
Article CAS PubMed Google Scholar
Rinner, O. et al. Identification of cross-linked peptides from large sequence databases. Nat. Methods 5, 315–318 (2008).
Article CAS PubMed PubMed Central Google Scholar
Walzthoeni, T. et al. False discovery rate estimation for cross-linked peptides identified by mass spectrometry. Nat. Methods 9, 901–903 (2012).
Article CAS PubMed Google Scholar
Bers, D. M., Patton, C. W. & Nuccitelli, R. A practical guide to the preparation of Ca(2+) buffers. Methods Cell. Biol. 99, 1–26 (2010).
Article CAS PubMed Google Scholar
Battye, T. G., Kontogiannis, L., Johnson, O., Powell, H. R. & Leslie, A. G. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr. D Biol. Crystallogr. 67, 271–281 (2011).
Article CAS PubMed PubMed Central Google Scholar
Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution? Acta Crystallogr. D Biol. Crystallogr. 69, 1204–1214 (2013).
Article CAS PubMed PubMed Central Google Scholar
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Article CAS PubMed PubMed Central Google Scholar
Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).
Article CAS PubMed Google Scholar
Cole, C., Barber, J. D. & Barton, G. J. The Jpred 3 secondary structure prediction server. Nucleic Acids Res. 36, W197–W201 (2008).
Article CAS PubMed PubMed Central Google Scholar
Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).
Article CAS PubMed PubMed Central Google Scholar
Yang, Z. et al. UCSF Chimera, MODELLER, and IMP: an integrated modeling system. J. Struct. Biol. 179, 269–278 (2012).
Article CAS PubMed Google Scholar
Holm, L. & Rosenstrom, P. Dali server: conservation mapping in 3D. Nucleic Acids Res. 38, W545–W549 (2010).
Article CAS PubMed PubMed Central Google Scholar
Obenauer, J. C., Cantley, L. C. & Yaffe, M. B. Scansite 2.0: Proteome-wide prediction of cell signaling interactions using short sequence motifs. Nucleic Acids Res. 31, 3635–3641 (2003).
Article CAS PubMed PubMed Central Google Scholar
Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).
Article CAS PubMed PubMed Central Google Scholar

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Acknowledgements

We thank Ambrose Cole, Duncan Laverty and staff at beamline I24 at the Diamond Light Source for help with data collection; Kanchan Chargar for assistance with cell culture; and Stuart Martin for help calibrating the plate reader for alphascreen and FRET recordings. MGG is a Wellcome Trust and Royal Society Sir Henry Dale fellow (104194/Z/14/Z), and receives support from the BBSRC (BB/N015274/1). F.S. is funded by the German Science Foundation Emmy Noether Programme (STE 2517/1-1) and is grateful for support from the DFG Collaborative Research Center (SFB) 969 and the Wellcome Trust (Grant 095951). R.A. was supported by ERC AdG-670821 (Proteomics 4D).

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Authors and Affiliations

Department of Neuroscience, Physiology & Pharmacology, University College London, Gower Street, London, WC1E 6BT, UK
Neha Patel & Matthew G. Gold
Department of Biology, University of Konstanz, Universitätsstrasse 10, 78457, Konstanz, Germany
Florian Stengel
Department of Biology, Institute of Molecular Systems Biology, ETH Zürich, 8093, Zürich, Switzerland
Florian Stengel & Ruedi Aebersold
Faculty of Science, University of Zürich, Zürich, Switzerland
Ruedi Aebersold

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Neha Patel
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Florian Stengel
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Ruedi Aebersold
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Matthew G. Gold
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Contributions

M.G.G. directed the project, performed alphascreen, phosphatase and pull-down assays, collected the X-ray diffraction data, and solved and analyzed the structure. N.P.: Prepared purified proteins, performed pull-down assays, and crystallized the complex. F.S.: Performed mass spectrometry experiments and processed the XL-MS data. R.A.: Supervised XL-MS experiments. M.G.G.: Prepared the manuscript with input from all the authors.

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Correspondence to Matthew G. Gold.

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Patel, N., Stengel, F., Aebersold, R. et al. Molecular basis of AKAP79 regulation by calmodulin. Nat Commun 8, 1681 (2017). https://doi.org/10.1038/s41467-017-01715-w

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Received: 24 January 2017
Accepted: 11 October 2017
Published: 22 November 2017
DOI: https://doi.org/10.1038/s41467-017-01715-w

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