Design and characterization of a protein fold switching network

Abstract

To better understand how amino acid sequence encodes protein structure, we engineered mutational pathways that connect three common folds (3α, β−grasp, and α/β−plait). The structures of proteins at high sequence-identity intersections in the pathways (nodes) were determined using NMR spectroscopy and analyzed for stability and function. To generate nodes, the amino acid sequence encoding a smaller fold is embedded in the structure of an ~50% larger fold and a new sequence compatible with two sets of native interactions is designed. This generates protein pairs with a 3α or β−grasp fold in the smaller form but an α/β−plait fold in the larger form. Further, embedding smaller antagonistic folds creates critical states in the larger folds such that single amino acid substitutions can switch both their fold and function. The results help explain the underlying ambiguity in the protein folding code and show that new protein structures can evolve via abrupt fold switching.

SSDraw: Software for generating comparative protein secondary structure diagrams

“…Because SSDraw uses the Matplotlib (Hunter, 2007) 2.2 | Advanced example 1: Comparing distinct structures with highly identical sequences using a custom color map SSDraw can be used to compare secondary structures of proteins with high levels of sequence identity but different folds (Figure 4). Extensive work has been performed to engineer (Alexander et al, 2007;Alexander et al, 2009;He et al, 2012;Ruan et al, 2023) and characterize (Sikosek et al, 2016;Solomon et al, 2023;Tian & Best, 2020) variants of the human serum albumin-binding protein GA and the immunoglobulin binding protein GB. While GA folds into a trihelical bundle, GB folds into a 4β + α structure.…”

Fluid protein fold space and its implications

“…Three lines of evidence suggest that protein fold space is fluid: (1) experimentally characterized amino acid sequences that interconvert between two stable folds with distinct secondary structures (fold-switching, [29] or metamorphic [27] proteins), (2) discoveries of short mutational pathways interconnecting proteins with distinct folds, [34,39,40] and (3) evolutionary analyses coupled with experiments demonstrating that fold switching is conserved among diverse homologs. [36,37] Demonstrating the first line of evidence, Figure 1B shows two proteins that switch between distinct stable folds: XCL1 (green), a human chemokine, and the C-terminal domain (CTD) of RfaH (yellow) a bacterial transcription factor.…”

“…The fluid fold space framework suggests additional protein evolution mechanisms: fold switching through stepwise mutation [39] and sequence elongation. [40,78] Single amino acid substitutions can switch an engineered human-serum-albumin (HSA)-binding 3-α-helical bundle protein to an immunoglobulin G (IgG)-binding α/β-grasp fold [39] and back again. [43] Furthermore, an engineered 56-amino acid 3-α-helical bundle protein with HSA binding activity can be embedded into a 99amino acid α/β-plait fold with protease inhibitor function [40] ; one such variant can switch from the helical bundle to the α/β-plait in response to temperature increases.…”

“…[40,78] Single amino acid substitutions can switch an engineered human-serum-albumin (HSA)-binding 3-α-helical bundle protein to an immunoglobulin G (IgG)-binding α/β-grasp fold [39] and back again. [43] Furthermore, an engineered 56-amino acid 3-α-helical bundle protein with HSA binding activity can be embedded into a 99amino acid α/β-plait fold with protease inhibitor function [40] ; one such variant can switch from the helical bundle to the α/β-plait in response to temperature increases. [79] Thus, both amino acid substitutions and sequence extensions can completely transform a protein's fold and function.…”

SSDraw: software for generating comparative protein secondary structure diagrams

“…This shortcoming especially impacts homologous proteins with non-conserved structural features arising from insertions, deletions, or mutations that cause substantial changes in secondary structure. Indeed, the need for easily interpretable structure diagrams is underscored by several recent studies highlighting how protein structure can transform dramatically in response to seemingly minor sequence changes [13][14][15][16][17] . To observe these transformations accurately, secondary structures of the proteins of interest must be registered, meaning that amino acids with annotated secondary structures must be aligned with their corresponding amino acids in a multiple sequence alignment (MSA).…”