Elucidation of transient protein-protein interactions within carrier protein-dependent biosynthesis

Fatty acid biosynthesis (FAB) is an essential and highly conserved metabolic pathway. In bacteria, this process is mediated by an elaborate network of protein•protein interactions (PPIs) involving a small, dynamic acyl carrier protein that interacts with dozens of other partner proteins (PPs). These PPIs have remained poorly characterized due to their dynamic and transient nature. Using a combination of solution-phase NMR spectroscopy and protein-protein docking simulations, we report a comprehensive residue-by-residue comparison of the PPIs formed during FAB in Escherichia coli. This technique describes and compares the molecular basis of six discrete binding events responsible for E. coli FAB and offers insights into a method to characterize these events and those in related carrier protein-dependent pathways.


Supplementary Materials for
Elucidation of transient protein-protein interactions within carrier protein-dependent biosynthesis Thomas G. Bartholow 1 , Terra Sztain 1 , Ashay Patel 1 , D. John Lee 1,3 , Megan A. Young 1 , Ruben Abagyan 2 , Michael D. Burkart 1* *Corresponding author. Email: mburkart@ucsd.edu Purification of FabF S10 Purification of TesA S11 Purification of AcpP S12 Titan peak fitting of FabF titration data S13 Titan peak fitting of FabG titration data S14 Titan peak fitting of FabI titration data S15 Titan peak fitting of TesA titration data Supplementary Tables S1 Interface and global alignment scores for docked models S2 Table of Figure S1. 1 H-15 N HSQC titration of C8-AcpP with FabI A) 5 overlayed 1 H-15 N HSQC spectra of 15 N-C8-AcpP titrated with increasing concentrations of unlabeled FabI. B) Bar chart of each AcpP residue's CSP with 4.0 molar equivalents of FabI. The mean is shown as a solid line and one standard deviation is the dashed line above. CSPs greater than this value are shown in colors as described by the insert. C) Two examples of residues displaying "titration curves," zoomed in from the boxed residues on the full spectra.

Figure S2 1 H-15 N HSQC titration of C8-AcpP with FabG
A) 5 overlayed 1 H-15 N HSQC spectra of 15 N-C8-AcpP titrated with increasing concentrations of unlabeled FabG. B) Bar chart of each AcpP residue's CSP with 6.0 molar equivalents of FabG, The mean is shown as a solid line and one standard deviation is the dashed line above. CSPs greater than this value are shown in colors as described by the insert. C) Two examples of residues displaying "titration curves," zoomed in from the boxed residues on the full spectra.

Figure S5
Interfaces of each partner studied. Two orientations of the interface of each enzyme studied in this work. Partner proteins are showed in gray with the AcpP in light purple. Negatively charged residues at the interface are colored red with positively charged residues showed in blue. Hydrophobic residues on the AcpP which lie at the interface are colored yellow while hydrophobic residues on the partner protein are tan. Figure S6 Residue assignments of C8-AcpP. Assignments were taken from a previous study performed in the Burkart lab 31 and used to assign the four titrations performed. Figure S7 Purification of FabG. FabG was purified as described in the methods. A 12% acrylamide denaturing SDS gel was run on the primary peak identified by the FPLC to verify the identity of the FabG.

Figure S8
Purification of FabI. FabI was purified by the standard method described in the methods. After FPLCing samples were chosen to run on an SDS gel to verify the purity of the protein. A 12% acrylamide denaturing SDS gel was run, showing very large quantities of protein purified for the experiment. Fractions 12, 13, and 14 were taken as the center of the peak and due to the high concentration of protein already present.

Figure S9
Purification of FabF. FabF was purified as described in the methods. The first peak on the FPLC was identified as the likely peak of interest. A sample of nickel purified FabF before the FPLC purification was run on the gel as a standard. A 12% polyacrylamide denaturing SDS gel was run in order to identify the useable fractions. Fractions A8-A12 were identified as sufficiently clean to concentrate for the experiment.

Figure S10
Purification of TesA. A/B) TesA was purified as described in the methods. The primary peak was sampled in order to ensure that the sample used was pure. A 12% polyacrylamide denaturing SDS gel was run focusing on the fractions making up the peak. Fractions 57, 58, 59, and 60 were collected and concentrated for the titration experiment. C) A test purification prior to the NMR experiment. This PAGE gel displayed an unsmeared example of the results expected for a TesA purification.

Figure S11
Purification of AcpP. An example of the purification of AcpP before an NMR experiment. Octanoyl-loaded AcpP was purified fresh for each titration. A 12% acrylamide denaturing SDS gel is shown. AcpP has very little absorbance at 280nm, as such the absorbance at 214 is also shown in magenta to help locate the AcpP's fractions. AcpP can be seen in fractions 19, 20, and 21, characteristically it travels unusually high on an SDS gel.
Figure S12 Titan peak fitting of FabF titration data. The FabF spectra and models were exported from the TITAN analysis program directly. Analysis was performed using the flexible docking method. Error analysis was performed using 300 steps of bootstrap error analysis. There was signal loss in the FabF experiment, likely due to the instability of the FabF protein and the presence of crashed partner protein in the titration.

Figure S13
Titan peak fitting of FabG titration data. The FabG spectra and models were exported from the TITAN analysis program directly. Analysis was performed using the flexible docking method. Error analysis was performed using 300 steps of bootstrap error analysis.
Figure S14 Titan peak fitting of FabI titration data. The FabI spectra and models were exported from the TITAN analysis program directly. Analysis was performed using the flexible docking method. Error analysis was performed using 300 steps of bootstrap error analysis Figure S15 Titan peak fitting of TesA titration data. The TesA spectra and models were exported from the TITAN analysis program directly. Analysis was performed using the flexible docking method. Error analysis was performed using 300 steps of bootstrap error analysis Table S1 Interface and global alignment scores for docked models. Alignments are generated from ICM docked models and aligned to crosslinked structures. The "informed" ACP residues are supplied form NMR titration data and the "informed" partner residues were taken from published data on essential electrostatic residues when possible.

Partner
Interface alignment (Å)      Table S7: Comparisons of the residue informed and uninformed top 10 docking poses. The 10 most energetically favorable poses from otherwise identical "informed" and "uninformed" docking calculations.