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1. Epistasis Creates Invariant Sites and Modulates the Rate of Molecular Evolution

   https://doi.org/10.1093/molbev/msac106  

   Patel, Ravi; Carnevale, Vincenzo; Kumar, Sudhir (May 2022, Molecular Biology and Evolution)

   Ozkan, Banu (Ed.)

   Abstract Invariant sites are a common feature of amino acid sequence evolution. The presence of invariant sites is frequently attributed to the need to preserve function through site-specific conservation of amino acid residues. Amino acid substitution models without a provision for invariant sites often fit the data significantly worse than those that allow for an excess of invariant sites beyond those predicted by models that only incorporate rate variation among sites (e.g., a Gamma distribution). An alternative is epistasis between sites to preserve residue interactions that can create invariant sites. Through computer-simulated sequence evolution, we evaluated the relative effects of site-specific preferences and site-site couplings in the generation of invariant sites and the modulation of the rate of molecular evolution. In an analysis of ten major families of protein domains with diverse sequence and functional properties, we find that the negative selection imposed by epistasis creates many more invariant sites than site-specific residue preferences alone. Further, epistasis plays an increasingly larger role in creating invariant sites over longer evolutionary periods. Epistasis also dictates rates of domain evolution over time by exerting significant additional purifying selection to preserve site couplings. These patterns illuminate the mechanistic role of epistasis in the processes underlying observed site invariance and evolutionary rates. 

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

   https://doi.org/10.1371/journal.pcbi.1010006  

   Ose, Nicholas J.; Butler, Brandon M.; Kumar, Avishek; Kazan, I. Can; Sanderford, Maxwell; Kumar, Sudhir; Ozkan, S. Banu (April 2022, PLOS Computational Biology)

   Wallqvist, Anders (Ed.)

   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 β-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. 

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3. Entrenchment and contingency in neutral protein evolution with epistasis

   https://doi.org/10.1101/2025.01.09.632266  

   Schmelkin, Lisa; Carnevale, Vincenzo; Haldane, Allan; Townsend, Jeffrey P; Chung, Sarah; Levy, Ronald M; Kumar, Sudhir (January 2025, bioRxiv)

   Abstract Protein sequence evolution in the presence of epistasis makes many previously acceptable amino acid residues at a site unfavorable over time. This phenomenon of entrenchment has also been observed with neutral substitutions using Potts Hamiltonian models. Here, we show that simulations using these models often evolve non-neutral proteins. We introduce a Neutral-with-Epistasis (N×E) model that incorporates purifying selection to conserve fitness, a requirement of neutral evolution. N×E protein evolution revealed a surprising lack of entrenchment, with site-specific amino-acid preferences remaining remarkably conserved, in biologically realistic time frames despite extensive residue coupling. Moreover, we found that the overdispersion of the molecular clock is caused by rate variation across sites introduced by epistasis in individual lineages, rather than by historical contingency. Therefore, substitutional entrenchment and rate contingency may indicate that adaptive and other non-neutral evolutionary processes were at play during protein evolution. 

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4. The Role of Conformational Dynamics and Allostery in Modulating Protein Evolution

   https://doi.org/10.1146/annurev-biophys-052118-115517  

   Campitelli, Paul; Modi, Tushar; Kumar, Sudhir; Ozkan, S. Banu (May 2020, Annual Review of Biophysics)

   Advances in sequencing techniques and statistical methods have made it possible not only to predict sequences of ancestral proteins but also to identify thousands of mutations in the human exome, some of which are disease associated. These developments have motivated numerous theories and raised many questions regarding the fundamental principles behind protein evolution, which have been traditionally investigated horizontally using the tip of the phylogenetic tree through comparative studies of extant proteins within a family. In this article, we review a vertical comparison of the modern and resurrected ancestral proteins. We focus mainly on the dynamical properties responsible for a protein's ability to adapt new functions in response to environmental changes. Using the Dynamic Flexibility Index and the Dynamic Coupling Index to quantify the relative flexibility and dynamic coupling at a site-specific, single-amino-acid level, we provide evidence that the migration of hinges, which are often functionally critical rigid sites, is a mechanism through which proteins can rapidly evolve. Additionally, we show that disease-associated mutations in proteins often result in flexibility changes even at positions distal from mutational sites, particularly in the modulation of active site dynamics. 

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5. Na+,K+-ATPase with Disrupted Na+ Binding Sites I and III Binds Na+ with Increased Affinity at Site II and Undergoes Na+-Activated Phosphorylation with ATP

   https://doi.org/10.3390/biom14010135  

   Nielsen, Hang N.; Holm, Rikke; Sweazey, Ryan; Andersen, Jens Peter; Artigas, Pablo; Vilsen, Bente (January 2024, Biomolecules)

   Na+,K+-ATPase actively extrudes three cytoplasmic Na+ ions in exchange for two extracellular K+ ions for each ATP hydrolyzed. The atomic structure with bound Na+ identifies three Na+ sites, named I, II, and III. It has been proposed that site III is the first to be occupied and site II last, when Na+ binds from the cytoplasmic side. It is usually assumed that the occupation of all three Na+ sites is obligatory for the activation of phosphoryl transfer from ATP. To obtain more insight into the individual roles of the ion-binding sites, we have analyzed a series of seven mutants with substitution of the critical ion-binding residue Ser777, which is a shared ligand between Na+ sites I and III. Surprisingly, mutants with large and bulky substituents expected to prevent or profoundly disturb Na+ access to sites I and III retain the ability to form a phosphoenzyme from ATP, even with increased apparent Na+ affinity. This indicates that Na+ binding solely at site II is sufficient to promote phosphorylation. These mutations appear to lock the membrane sector into an E1-like configuration, allowing Na+ but not K+ to bind at site II, while the cytoplasmic sector undergoes conformational changes uncoupled from the membrane sector. 

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