Comprehensive analysis of loops at protein-protein interfaces for macrocycle design

Journal name:
Nature Chemical Biology
Volume:
10,
Pages:
716–722
Year published:
DOI:
doi:10.1038/nchembio.1580
Received
Accepted
Published online

Abstract

Inhibiting protein-protein interactions (PPIs) with synthetic molecules remains a frontier of chemical biology. Many PPIs have been successfully targeted by mimicking α-helices at interfaces, but most PPIs are mediated by nonhelical, nonstrand peptide loops. We sought to comprehensively identify and analyze these loop-mediated PPIs by writing and implementing LoopFinder, a customizable program that can identify loop-mediated PPIs within all of the protein-protein complexes in the Protein Data Bank. Comprehensive analysis of the entire set of 25,005 interface loops revealed common structural motifs and unique features that distinguish loop-mediated PPIs from other PPIs. 'Hot loops', named in analogy to protein hot spots, were identified as loops with favorable properties for mimicry using synthetic molecules. The hot loops and their binding partners represent new and promising PPIs for the development of macrocycle and constrained peptide inhibitors.

At a glance

Figures

  1. Identification of hot loops.
    Figure 1: Identification of hot loops.

    Hot loops are identified as those loops that satisfy one or more of three criteria: the average ΔΔGresidue over the entire loop is greater than 1 kcal mol−1, the loop has three or more hot-spot residues (ΔΔGresidue ≥ 1 kcal mol−1) and the loop has two or more consecutive hot-spot residues. Representative loops that satisfy each of these criteria are shown within the blue, red and yellow circles (structures with PDB codes 1AXI, 1GK9 and 1L2U, respectively). Some hot loops satisfy two of these criteria, with representative loops from these categories shown in the purple, orange and green boxes (2QNR, 1GK9 and 2FPF, respectively). In addition, 67 hot loops satisfy all three criteria, an example of which is shown in the gray box to the left (PDB code 2AST). All structures, rendered in PyMOL42, show the chain at the interface in blue, the binding partner as a gray surface, the hot loop in green and hot spots in orange (ΔΔGresidue ≥ 1 kcal mol−1) and yellow (ΔΔGresidue ≥ 2 kcal mol−1). Representative hot loops display a wide range of loop structures and modes of interaction with the partner surface.

  2. Visualization of different loop structures observed among the hot loops.
    Figure 2: Visualization of different loop structures observed among the hot loops.

    Representative examples of each type of loop are shown within each circle, including β-turns (PDB code 2ZZC); Schellman loops (PDB code 2OL1); αβ-motifs (PDB code 2DVT); β-bulges (PDB code 3GBT); β-hairpins (PDB code 1T3I); Asx turns and motifs (PDB code 1LIA); S/T-turns, motifs and staples (PDB code 1Y1X); and γ-turns (PDB code 2IX5). The remaining two categories shown above are α-helical regions identified by their backbone torsional angles (PDB code 2BM8) and loops lacking canonical structural motifs (PDB code 3KYH). All structures, rendered in PyMOL42, show the hot loop in green and hot spots in orange (ΔΔGresidue ≥ 1 kcal mol−1) or yellow (ΔΔGresidue ≥ 2 kcal mol−1).

  3. Interface loops use a unique set of amino acids to recognize their binding partners.
    Figure 3: Interface loops use a unique set of amino acids to recognize their binding partners.

    The percent abundance values of each amino acid were normalized relative to propensity to reside on a protein surface20. These normalized values were further broken down into all residues (blue), hot spot residues (purple) and non-hot-spot residues (green).

  4. Established and new targets for inhibitor design.
    Figure 4: Established and new targets for inhibitor design.

    (a) LoopFinder identified a hot loop on the surface of ​hGH that is known to be essential for binding of ​hGHbp (PDB code 1HWG)24. (b) Hot loop within the transcription factor ​Nrf2 that binds its repressor, ​Keap1 (PDB code 2FLU)26. (c) The sC-connector loop of ​TIMP-3 is a hot loop that binds the S2 pocket of ​TACE (PDB code 3CKI)43. (d) The interaction between ​Skp2 and ​Cks1 is essential for the formation of the SCFSkp2 complex and its ubiquitin E3 ligase activity (PDB code 2AST)31. (e) Inhibition of the histone acetyltransferase (HAT) MSL complex is a nw target identified by LoopFinder (PDB code 2Y0N)37. All structures, rendered in PyMOL42, show the hot loop in green and hot spots in orange (ΔΔGresidue ≥ 1 kcal mol−1) or yellow (ΔΔGresidue ≥ 2 kcal mol−1).

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Author information

  1. These authors contributed equally to this work.

    • Jason Gavenonis,
    • Bradley A Sheneman &
    • Timothy R Siegert

Affiliations

  1. Department of Chemistry, Tufts University, Medford, Massachusetts, USA.

    • Jason Gavenonis,
    • Bradley A Sheneman,
    • Timothy R Siegert,
    • Matthew R Eshelman &
    • Joshua A Kritzer

Contributions

B.A.S. wrote the LoopFinder code. J.G., B.A.S. and J.A.K. performed troubleshooting and debugged and parameterized Loopfinder and Rosetta-based computational alanine scanning. J.G., T.R.S., M.R.E. and J.A.K. analyzed and contextualized data. J.G., T.R.S. and J.A.K. produced figures and tables and wrote the paper.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

PDF files

  1. Supplementary Text and Figures (1,744 KB)

    Supplementary Note, Supplementary Results, Supplementary Figures 1–8 and Supplementary Tables 1–6.

Excel files

  1. Supplementary Data Set 1 (315 KB)

    The entire set of hot loops generated by LoopFinder.

  2. Supplementary Data Set 2 (51 KB)

    The subset of 364 hot loops that do not contain two or more consecutive hot spots.

Additional data