Some mechanistic underpinnings of molecular adaptations of SARS-COV-2 spike protein by integrating candidate adaptive polymorphisms with protein dynamics.

We integrate evolutionary predictions based on the neutral theory of molecular evolution with protein dynamics to generate mechanistic insight into the molecular adaptations of the SARS-COV-2 spike (S) protein. With this approach, we first identified candidate adaptive polymorphisms (CAPs) of the SARS-CoV-2 S protein and assessed the impact of these CAPs through dynamics analysis. Not only have we found that CAPs frequently overlap with well-known functional sites, but also, using several different dynamics-based metrics, we reveal the critical allosteric interplay between SARS-CoV-2 CAPs and the S protein binding sites with the human ACE2 (hACE2) protein. CAPs interact far differently with the hACE2 binding site residues in the open conformation of the S protein compared to the closed form. In particular, the CAP sites control the dynamics of binding residues in the open state, suggesting an allosteric control of hACE2 binding. We also explored the characteristic mutations of different SARS-CoV-2 strains to find dynamic hallmarks and potential effects of future mutations. Our analyses reveal that Delta strain-specific variants have non-additive (i.e., epistatic) interactions with CAP sites, whereas the less pathogenic Omicron strains have mostly additive mutations. Finally, our dynamics-based analysis suggests that the novel mutations observed in the Omicron strain epistatically interact with the CAP sites to help escape antibody binding.

Engineering gain-of-function mutants of a WW domain by dynamics and structural analysis.

Proteins gain optimal fitness such as foldability and function through evolutionary selection. However, classical studies have found that evolutionarily designed protein sequences alone cannot guarantee foldability, or at least not without considering local contacts associated with the initial folding steps. We previously showed that foldability and function can be restored by removing frustration in the folding energy landscape of a model WW domain protein, CC16, which was designed based on Statistical Coupling Analysis (SCA). Substitutions ensuring the formation of five local contacts identified as "on-path" were selected using the closest homolog native folded sequence, N21. Surprisingly, the resulting sequence, CC16-N21, bound to Group I peptides, while N21 did not. Here, we identified single-point mutations that enable N21 to bind a Group I peptide ligand through structure and dynamic-based computational design. Comparison of the docked position of the CC16-N21/ligand complex with the N21 structure showed that residues at positions 9 and 19 are important for peptide binding, whereas the dynamic profiles identified position 10 as allosterically coupled to the binding site and exhibiting different dynamics between N21 and CC16-N21. We found that swapping these positions in N21 with matched residues from CC16-N21 recovers nature-like binding affinity to N21. This study validates the use of dynamic profiles as guiding principles for affecting the binding affinity of small proteins.

Allosteric regulatory control in dihydrofolate reductase is revealed by dynamic asymmetry.

We investigated the relationship between mutations and dynamics in Escherichia coli dihydrofolate reductase (DHFR) using computational methods. Our study focused on the M20 and FG loops, which are known to be functionally important and affected by mutations distal to the loops. We used molecular dynamics simulations and developed position-specific metrics, including the dynamic flexibility index (DFI) and dynamic coupling index (DCI), to analyze the dynamics of wild-type DHFR and compared our results with existing deep mutational scanning data. Our analysis showed a statistically significant association between DFI and mutational tolerance of the DHFR positions, indicating that DFI can predict functionally beneficial or detrimental substitutions. We also applied an asymmetric version of our DCI metric (DCIasym ) to DHFR and found that certain distal residues control the dynamics of the M20 and FG loops, whereas others are controlled by them. Residues that are suggested to control the M20 and FG loops by our DCIasym metric are evolutionarily nonconserved; mutations at these sites can enhance enzyme activity. On the other hand, residues controlled by the loops are mostly deleterious to function when mutated and are also evolutionary conserved. Our results suggest that dynamics-based metrics can identify residues that explain the relationship between mutation and protein function or can be targeted to rationally engineer enzymes with enhanced activity.

Design of novel cyanovirin-N variants by modulation of binding dynamics through distal mutations.

We develop integrated co-evolution and dynamic coupling (ICDC) approach to identify, mutate, and assess distal sites to modulate function. We validate the approach first by analyzing the existing mutational fitness data of TEM-1 ß-lactamase and show that allosteric positions co-evolved and dynamically coupled with the active site significantly modulate function. We further apply ICDC approach to identify positions and their mutations that can modulate binding affinity in a lectin, cyanovirin-N (CV-N), that selectively binds to dimannose, and predict binding energies of its variants through Adaptive BP-Dock. Computational and experimental analyses reveal that binding enhancing mutants identified by ICDC impact the dynamics of the binding pocket, and show that rigidification of the binding residues compensates for the entropic cost of binding. This work suggests a mechanism by which distal mutations modulate function through dynamic allostery and provides a blueprint to identify candidates for mutagenesis in order to optimize protein function.

Investigating the allosteric response of the PICK1 PDZ domain to different ligands with all-atom simulations.

The PDZ family is comprised of small modular domains that play critical roles in the allosteric modulation of many cellular signaling processes by binding to the C-terminal tail of different proteins. As dominant modular proteins that interact with a diverse set of peptides, it is of particular interest to explore how different binding partners induce different allosteric effects on the same PDZ domain. Because the PICK1 PDZ domain can bind different types of ligands, it is an ideal test case to answer this question and explore the network of interactions that give rise to dynamic allostery. Here, we use all-atom molecular dynamics simulations to explore dynamic allostery in the PICK1 PDZ domain by modeling two PICK1 PDZ systems: PICK1 PDZ-DAT and PICK1 PDZ-GluR2. Our results suggest that ligand binding to the PICK1 PDZ domain induces dynamic allostery at the αA helix that is similar to what has been observed in other PDZ domains. We found that the PICK1 PDZ-ligand distance is directly correlated with both dynamic changes of the αA helix and the distance between the αA helix and ßB strand. Furthermore, our work identifies a hydrophobic core between DAT/GluR2 and I35 as a key interaction in inducing such dynamic allostery. Finally, the unique interaction patterns between different binding partners and the PICK1 PDZ domain can induce unique dynamic changes to the PICK1 PDZ domain. We suspect that unique allosteric coupling patterns with different ligands may play a critical role in how PICK1 performs its biological functions in various signaling networks.

Dynamic coupling of residues within proteins as a mechanistic foundation of many enigmatic pathogenic missense variants.

Many pathogenic missense mutations are found in protein positions that are neither well-conserved nor fall in any known functional domains. Consequently, we lack any mechanistic underpinning of dysfunction caused by such mutations. We explored the disruption of allosteric dynamic coupling between these positions and the known functional sites as a possible mechanism for pathogenesis. In this study, we present an analysis of 591 pathogenic missense variants in 144 human enzymes that suggests that allosteric dynamic coupling of mutated positions with known active sites is a plausible biophysical mechanism and evidence of their functional importance. We illustrate this mechanism in a case study of ß-Glucocerebrosidase (GCase) in which a vast majority of 94 sites harboring Gaucher disease-associated missense variants are located some distance away from the active site. An analysis of the conformational dynamics of GCase suggests that mutations on these distal sites cause changes in the flexibility of active site residues despite their distance, indicating a dynamic communication network throughout the protein. The disruption of the long-distance dynamic coupling caused by missense mutations may provide a plausible general mechanistic explanation for biological dysfunction and disease.

The Role of Rigid Residues in Modulating TEM-1 ß-Lactamase Function and Thermostability.

The relationship between protein motions (i.e., dynamics) and enzymatic function has begun to be explored in ß-lactamases as a way to advance our understanding of these proteins. In a recent study, we analyzed the dynamic profiles of TEM-1 (a ubiquitous class A ß-lactamase) and several ancestrally reconstructed homologues. A chief finding of this work was that rigid residues that were allosterically coupled to the active site appeared to have profound effects on enzyme function, even when separated from the active site by many angstroms. In the present work, our aim was to further explore the implications of protein dynamics on ß-lactamase function by altering the dynamic profile of TEM-1 using computational protein design methods. The Rosetta software suite was used to mutate amino acids surrounding either rigid residues that are highly coupled to the active site or to flexible residues with no apparent communication with the active site. Experimental characterization of ten designed proteins indicated that alteration of residues surrounding rigid, highly coupled residues, substantially affected both enzymatic activity and stability; in contrast, native-like activities and stabilities were maintained when flexible, uncoupled residues, were targeted. Our results provide additional insight into the structure-function relationship present in the TEM family of ß-lactamases. Furthermore, the integration of computational protein design methods with analyses of protein dynamics represents a general approach that could be used to extend our understanding of the relationship between dynamics and function in other enzyme classes.

Author Correction: Plant-expressed cocaine hydrolase variants of butyrylcholinesterase exhibit altered allosteric effects of cholinesterase activity and increased inhibitor sensitivity.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Coevolving residues inform protein dynamics profiles and disease susceptibility of nSNVs.

The conformational dynamics of proteins is rarely used in methodologies used to predict the impact of genetic mutations due to the paucity of three-dimensional protein structures as compared to the vast number of available sequences. Until now a three-dimensional (3D) structure has been required to predict the conformational dynamics of a protein. We introduce an approach that estimates the conformational dynamics of a protein, without relying on structural information. This de novo approach utilizes coevolving residues identified from a multiple sequence alignment (MSA) using Potts models. These coevolving residues are used as contacts in a Gaussian network model (GNM) to obtain protein dynamics. B-factors calculated using sequence-based GNM (Seq-GNM) are in agreement with crystallographic B-factors as well as theoretical B-factors from the original GNM that utilizes the 3D structure. Moreover, we demonstrate the ability of the calculated B-factors from the Seq-GNM approach to discriminate genomic variants according to their phenotypes for a wide range of proteins. These results suggest that protein dynamics can be approximated based on sequence information alone, making it possible to assess the phenotypes of nSNVs in cases where a 3D structure is unknown. We hope this work will promote the use of dynamics information in genetic disease prediction at scale by circumventing the need for 3D structures.

Plant-expressed cocaine hydrolase variants of butyrylcholinesterase exhibit altered allosteric effects of cholinesterase activity and increased inhibitor sensitivity.

Butyrylcholinesterase (BChE) is an enzyme with broad substrate and ligand specificities and may function as a generalized bioscavenger by binding and/or hydrolyzing various xenobiotic agents and toxicants, many of which target the central and peripheral nervous systems. Variants of BChE were rationally designed to increase the enzyme's ability to hydrolyze the psychoactive enantiomer of cocaine. These variants were cloned, and then expressed using the magnICON transient expression system in plants and their enzymatic properties were investigated. In particular, we explored the effects that these site-directed mutations have over the enzyme kinetics with various substrates of BChE. We further compared the affinity of various anticholinesterases including organophosphorous nerve agents and pesticides toward these BChE variants relative to the wild type enzyme. In addition to serving as a therapy for cocaine addiction-related diseases, enhanced bioscavenging against other harmful agents could add to the practicality and versatility of the plant-derived recombinant enzyme as a multivalent therapeutic.