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

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.

Figure 1

Identification of hot loops. Hot loops are identified as those loops that satisfy one or more of three criteria: the average ΔΔG_residue over the entire loop is greater than 1 kcal/mol, the loop has three or more hot spot residues (ΔΔG_residue ≥ 1 kcal/mol), 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 from 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 (2AST). All structures, rendered in Pymol, 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 (ΔΔG_residue ≥ 1 kcal/mol) and yellow (ΔΔG_residue ≥ 2 kcal/mol). Representative hot loops display a wide range of loop structures and modes of interaction with the partner surface.

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

Figure 3

Interface loops use a unique set of amino acids to recognize their binding partners. The percent abundances of each amino acid were normalized relative to propensity to reside on a protein surface. These normalized values were further broken down into all residues (blue), hot spot residues (red) and non-hot spot residues (green).

Figure 4

Established and novel 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 (1HWG). b) Hot loop within the transcription factor Nrf2 that binds its repressor, Keap1 (2FLU). c) The sC-connector loop of TIMP-3 is a hot loop that binds to the S2 pocket of TACE (3CKI). d) The interaction between Skp2 and Cks1 is essential for the formation of the SCF^Skp2 complex and its ubiquitin E3 ligase activity (2AST). e) Inhibition of the histone acetyltransferase (HAT) MSL complex is a novel target identified by LoopFinder (2Y0N). All structures, rendered in Pymol, show the hot loop in green, and hot spots in orange (ΔΔG_residue ≥ 1 kcal/mol) or yellow (ΔΔG_residue ≥ 2 kcal/mol).