Back in time to the Gly-rich prototype of the phosphate binding elementary function.

Binding of nucleotides and their derivatives is one of the most ancient elementary functions dating back to the Origin of Life. We review here the works considering one of the key elements in binding of (di)nucleotide-containing ligands - phosphate binding. We start from a brief discussion of major participants, conditions, and events in prebiotic evolution that resulted in the Origin of Life. Tracing back to the basic functions, including metal and phosphate binding, and, potentially, formation of primitive protein-protein interactions, we focus here on the phosphate binding. Critically assessing works on the structural, functional, and evolutionary aspects of phosphate binding, we perform a simple computational experiment reconstructing its most ancient and generic sequence prototype. The profiles of the phosphate binding signatures have been derived in form of position-specific scoring matrices (PSSMs), their peculiarities depending on the type of the ligands have been analyzed, and evolutionary connections between them have been delineated. Then, the apparent prototype that gave rise to all relevant phosphate-binding signatures had also been reconstructed. We show that two major signatures of the phosphate binding that discriminate between the binding of dinucleotide- and nucleotide-containing ligands are GxGxxG and GxxGxG, respectively. It appears that the signature archetypal for dinucleotide-containing ligands is more generic, and it can frequently bind phosphate groups in nucleotide-containing ligands as well. The reconstructed prototype's key signature GxGGxG underlies the role of glycine residues in providing flexibility and interactions necessary for binding the phosphate groups. The prototype also contains other ancient amino acids, valine, and alanine, showing versatility towards evolutionary design and functional diversification.

Living in trinity of extremes: Genomic and proteomic signatures of halophilic, thermophilic, and pH adaptation.

Since nucleic acids and proteins of unicellular prokaryotes are directly exposed to extreme environmental conditions, it is possible to explore the genomic-proteomic compositional determinants of molecular mechanisms of adaptation developed by them in response to harsh environmental conditions. Using a wealth of currently available complete genomes/proteomes we were able to explore signatures of adaptation to three environmental factors, pH, salinity, and temperature, observing major trends in compositions of their nucleic acids and proteins. We derived predictors of thermostability, halophilic, and pH adaptations and complemented them by the principal components analysis. We observed a clear difference between thermophilic and salinity/pH adaptations, whereas latter invoke seemingly overlapping mechanisms. The genome-proteome compositional trade-off reveals an intricate balance between the work of base paring and base stacking in stabilization of coding DNA and r/tRNAs, and, at the same time, universal requirements for the stability and foldability of proteins regardless of the nucleotide biases. Nevertheless, we still found hidden fingerprints of ancient evolutionary connections between the nucleotide and amino acid compositions indicating their emergence, mutual evolution, and adjustment. The evolutionary perspective on the adaptation mechanisms is further studied here by means of the comparative analysis of genomic/proteomic traits of archaeal and bacterial species. The overall picture of genomic/proteomic signals of adaptation obtained here provides a foundation for future engineering and design of functional biomolecules resistant to harsh environments.

Allosteric drugs: New principles and design approaches.

Focusing on an important biomedical implication of allostery - design of allosteric drugs, we describe characteristics of allosteric sites, effectors, and their modes of actions distinguishing them from the orthosteric counterparts and calling for new principles and protocols in the quests for allosteric drugs. We show the importance of considering both binding affinity and allosteric signaling in establishing the structure-activity relationships (SARs) toward design of allosteric effectors, arguing that pairs of allosteric sites and their effector ligands - the site-effector pairs - should be generated and adjusted simultaneously in the framework of what we call directed design protocol. Key ideas and approaches for designing allosteric effectors including reverse perturbation, targeted and agnostic analysis are also discussed here. Several promising computational approaches are highlighted, along with the need for and potential advantages of utilizing generative models to facilitate discovery/design of new allosteric drugs.

Disrupted chromatin architecture in olfactory sensory neurons: looking for the link from COVID-19 infection to anosmia.

We tackle here genomic mechanisms of a rapid onset and recovery from anosmia-a potential diagnostic indicator for early-stage COVID-19 infection. Based on previous observations on how olfactory receptor (OR) gene expression is regulated via chromatin structure in mice, we hypothesized that the disruption of the OR gene expression and, respectively, deficiency of the OR function can be caused by chromatin reorganization taking place upon SARS-CoV-2 infection. We obtained chromatin ensemble reconstructions from COVID-19 patients and control samples using our original computational framework for the whole-genome 3D chromatin ensemble reconstruction. Specifically, we used megabase-scale structural units and effective interactions between them obtained in the Markov State modelling of the Hi-C contact network as an unput in the stochastic embedding procedure of the whole-genome 3D chromatin ensemble reconstruction. We have also developed here a new procedure for analyzing fine structural hierarchy with (sub)TAD-size units in local chromatin regions, which we apply here to parts of chromosomes containing OR genes and corresponding regulatory elements. We observed structural modifications in COVID-19 patients on different levels of chromatin organization, from the alteration of whole genome structure and chromosomal intermingling to reorganization of contacts between chromatin loops at the level of topologically associating domains. While complementary data on known regulatory elements point to potential pathology-associated changes within the overall picture of chromatin alterations, further investigation using additional epigenetic factors mapped on 3D reconstructions with improved resolution will be required for better understanding of anosmia caused by SARS-CoV-2 infection.

Sequence-dependent model of allosteric communication.

The omnipresence and diversity of allosteric regulation in proteins and protein associations complemented by the potential for the design of allosterically acting biologics and drugs call for the development of a new generation of computational models for the analysis of allostery and rational engineering/design of desired signaling and effector molecules determining it. One of the most important challenges is the consideration of the role of amino acid sequence in forming the protein's allosteric communication, including the mode and strength of the allosteric signal that is communicated to the regulated functional site. Here, we present the network-based model with a sequence dependence added in consideration of allosteric communication by combining the structure-based statistical mechanical model of allostery with the Miyazawa-Jernigan residue-residue potential. Applying the model in the analysis of five classical allosteric proteins, we found that it is necessary to consider the following two major determinants: (i) the free energy exerted by the allosteric site on the regulated one and (ii) the background (average) change in dynamics of the overall structure. We show that working together these two components determine the allosteric modulation, calling one to study their dependence on structures, oligomerization states, and sequence divergence in different proteins.

AlloMAPS 2: allosteric fingerprints of the AlphaFold and Pfam-trRosetta predicted structures for engineering and design.

AlloMAPS 2 is an update of the Allosteric Mutation Analysis and Polymorphism of Signalling database, which contains data on allosteric communication obtained for predicted structures in the AlphaFold database (AFDB) and trRosetta-predicted Pfam domains. The data update contains Allosteric Signalling Maps (ASMs) and Allosteric Probing Maps (APMs) quantifying allosteric effects of mutations and of small probe binding, respectively. To ensure quality of the ASMs and APMs, we performed careful and accurate selection of protein sets containing high-quality predicted structures in both databases for each organism/structure, and the data is available for browsing and download. The data for remaining structures are available for download and should be used at user's discretion and responsibility. We believe these massive data can facilitate both diagnostics and drug design within the precision medicine paradigm. Specifically, it can be instrumental in the analysis of allosteric effects of pathological and rescue mutations, providing starting points for fragment-based design of allosteric effectors. The exhaustive character of allosteric signalling and probing fingerprints will be also useful in future developments of corresponding machine learning applications. The database is freely available at: http://allomaps.bii.a-star.edu.sg.

Learning About Allosteric Drugs and Ways to Design Them.

While the accelerating quest for precision medicine requires new individually targeting and selective drugs, and the ability to work with so-called undruggable targets, the realm of allosteric drugs meeting this need remains largely uncharted. Generalizing the observations on two major drug targets with widely observed inherent allostery, GPCRs and kinases, we describe and discuss basic allosteric modes of action that are universally applicable in all types of structures and functions. Using examples of Class A GPCRs and CMGC protein kinases, we show how Allosteric Signalling and Probing Fingerprints can be used to identify potential allosteric sites and reveal effector-leads that may serve as a starting point for the development of allosteric drugs targeting these regulatory sites. A set of distinct characteristics of allosteric ligands was established, which highlights the versatility of their design and make them advantageous before their orthosteric counterparts in personalized medicine. We argue that rational design of allosteric drugs should begin with the search for latent sites or design of non-natural binding sites followed by fragment-based design of allosteric ligands and by the mutual adjustment of the site-ligand pair in order to achieve required drug efficacy. On the basis of the perturbative nature and reversibility of allosteric communication, we propose a generic protocol for computational design of allosteric effectors, enabling also the allosteric tuning of biologics, in obtaining allosteric control over protein functions.

Conservation and Diversity in Allosteric Fingerprints of Proteins for Evolutionary-inspired Engineering and Design.

Hand-in-hand work of physics and evolution delivered protein universe with diversity of forms, sizes, and functions. Pervasiveness and advantageous traits of allostery made it an important component of the protein function regulation, calling for thorough investigation of its structural determinants and evolution. Learning directly from nature, we explored here allosteric communication in several major folds and repeat proteins, including α/ß and ß-barrels, ß-propellers, Ig-like fold, ankyrin and α/ß leucine-rich repeat proteins, which provide structural platforms for many different enzymatic and signalling functions. We obtained a picture of conserved allosteric communication characteristic in different fold types, modifications of the structure-driven signalling patterns via sequence-determined divergence to specific functions, as well as emergence and potential diversification of allosteric regulation in multi-domain proteins and oligomeric assemblies. Our observations will be instrumental in facilitating the engineering and de novo design of proteins with allosterically regulated functions, including development of therapeutic biologics. In particular, results described here may guide the identification of the optimal structural platforms (e.g. fold type, size, and oligomerization states) and the types of diversifications/perturbations, such as mutations, effector binding, and order-disorder transition. The tunable allosteric linkage across distant regions can be used as a pivotal component in the design/engineering of modular biological systems beyond the traditional scaffolding function.

Allosteric perspective on the mutability and druggability of the SARS-CoV-2 Spike protein.

Recent developments in the SARS-CoV-2 pandemic point to its inevitable transformation into an endemic disease, urging both refinement of diagnostics for emerging variants of concern (VOCs) and design of variant-specific drugs in addition to vaccine adjustments. Exploring the structure and dynamics of the SARS-CoV-2 Spike protein, we argue that the high-mutability characteristic of RNA viruses coupled with the remarkable flexibility and dynamics of viral proteins result in a substantial involvement of allosteric mechanisms. While allosteric effects of mutations should be considered in predictions and diagnostics of new VOCs, allosteric drugs advantageously avoid escape mutations via non-competitive inhibition originating from alternative distal locations. The exhaustive allosteric signaling and probing maps presented herein provide a comprehensive picture of allostery in the spike protein, making it possible to locate potential mutations that could work as new VOC "drivers" and to determine binding patches that may be targeted by newly developed allosteric drugs.

Exploring the Allosteric Territory of Protein Function.

While the pervasiveness of allostery in proteins is commonly accepted, we further show the generic nature of allosteric mechanisms by analyzing here transmembrane ion-channel viroporin 3a and RNA-dependent RNA polymerase (RdRp) from SARS-CoV-2 along with metabolic enzymes isocitrate dehydrogenase 1 (IDH1) and fumarate hydratase (FH) implicated in cancers. Using the previously developed structure-based statistical mechanical model of allostery (SBSMMA), we share our experience in analyzing the allosteric signaling, predicting latent allosteric sites, inducing and tuning targeted allosteric response, and exploring the allosteric effects of mutations. This, yet incomplete list of phenomenology, forms a complex and unique allosteric territory of protein function, which should be thoroughly explored. We propose a generic computational framework, which not only allows one to obtain a comprehensive allosteric control over proteins but also provides an opportunity to approach the fragment-based design of allosteric effectors and drug candidates. The advantages of allosteric drugs over traditional orthosteric compounds, complemented by the emerging role of the allosteric effects of mutations in the expansion of the cancer mutational landscape and in the increased mutability of viral proteins, leave no choice besides further extensive studies of allosteric mechanisms and their biomedical implications.

Three-dimensional chromatin ensemble reconstruction via stochastic embedding.

We propose a comprehensive method for reconstructing the whole-genome chromatin ensemble from the Hi-C data. The procedure starts from Markov state modeling (MSM), delineating the structural hierarchy of chromatin organization with partitioning and effective interactions archetypal for corresponding levels of hierarchy. The stochastic embedding procedure introduced in this work provides the 3D ensemble reconstruction, using effective interactions obtained by the MSM as the input. As a result, we obtain the structural ensemble of a genome, allowing one to model the functional and the cell-type variability in the chromatin structure. The whole-genome reconstructions performed on the human B lymphoblastoid (GM12878) and lung fibroblast (IMR90) Hi-C data unravel distinctions in their morphologies and in the spatial arrangement of intermingling chromosomal territories, paving the way to studies of chromatin dynamics, developmental changes, and conformational transitions taking place in normal cells and during potential pathological developments.

Deriving and Using Descriptors of Elementary Functions in Rational Protein Design.

The rational design of proteins with desired functions requires a comprehensive description of the functional building blocks. The evolutionary conserved functional units constitute nature's toolbox; however, they are not readily available to protein designers. This study focuses on protein units of subdomain size that possess structural properties and amino acid residues sufficient to carry out elementary reactions in the catalytic mechanisms. The interactions within such elementary functional loops (ELFs) and the interactions with the surrounding protein scaffolds constitute the descriptor of elementary function. The computational approach to deriving descriptors directly from protein sequences and structures and applying them in rational design was implemented in a proof-of-concept DEFINED-PROTEINS software package. Once the descriptor is obtained, the ELF can be fitted into existing or novel scaffolds to obtain the desired function. For instance, the descriptor may be used to determine the necessary spatial restraints in a fragment-based grafting protocol. We illustrated the approach by applying it to well-known cases of ELFs, including phosphate-binding P-loop, diphosphate-binding glycine-rich motif, and calcium-binding EF-hand motif, which could be used to jumpstart templates for user applications. The DEFINED-PROTEINS package is available for free at https://github.com/MelvinYin/Defined_Proteins.

Synergistic Allostery in Multiligand-Protein Interactions.

Amide hydrogen-deuterium exchange mass spectrometry is powerful for describing combinatorial coupling effects of a cooperative ligand pair binding at noncontiguous sites: adenosine at the ATP-pocket and a docking peptide (PIFtide) at the PIF-pocket, on a model protein kinase PDK1. Binding of two ligands to PDK1 reveal multiple hotspots of synergistic allostery with cumulative effects greater than the sum of individual effects mediated by each ligand. We quantified this synergism and ranked these hotspots using a difference in deuteration-based approach, which showed that the strongest synergistic effects were observed at three of the critical catalytic loci of kinases: the αB-αC helices, and HRD-motif loop, and DFG-motif. Additionally, we observed weaker synergistic effects at a distal GHI-subdomain locus. Synergistic changes in deuterium exchange observed at a distal site but not at the intermediate sites of the large lobe of the kinase reveals allosteric propagation in proteins to operate through two modes. Direct electrostatic interactions between polar and charged amino acids that mediate targeted relay of allosteric signals, and diffused relay of allosteric signals through soft matter-like hydrophobic core amino acids. Furthermore, we provide evidence that the conserved ß-3 strand lysine of protein kinases (Lys111 of PDK1) functions as an integrator node to coordinate allosteric coupling of the two ligand-binding sites. It maintains indirect interactions with the ATP-pocket and mediates a critical salt bridge with a glutamate (Glu130) of αC helix, which is conserved across all kinases. In summary, allosteric propagation in cooperative, dual-liganded enzyme targets is bidirectional and synergistic and offers a strategy for combinatorial drug development.

AlloSigMA 2: paving the way to designing allosteric effectors and to exploring allosteric effects of mutations.

The AlloSigMA 2 server provides an interactive platform for exploring the allosteric signaling caused by ligand binding and/or mutations, for analyzing the allosteric effects of mutations and for detecting potential cancer drivers and pathogenic nsSNPs. It can also be used for searching latent allosteric sites and for computationally designing allosteric effectors for these sites with required agonist/antagonist activity. The server is based on the implementation of the Structure-Based Statistical Mechanical Model of Allostery (SBSMMA), which allows one to evaluate the allosteric free energy as a result of the perturbation at per-residue resolution. The Allosteric Signaling Map (ASM) providing a comprehensive residue-by-residue allosteric control over the protein activity can be obtained for any structure of interest. The Allosteric Probing Map (APM), in turn, allows one to perform the fragment-based-like computational design experiment aimed at finding leads for potential allosteric effectors. The server can be instrumental in elucidating of allosteric mechanisms and actions of allosteric mutations, and in the efforts on design of new elements of allosteric control. The server is freely available at: http://allosigma.bii.a-star.edu.sg.

Allosteric drugs and mutations: chances, challenges, and necessity.

Allosteric drugs have become an indispensable toolbox of rapidly developing precision medicine, having already established reputation of advantages over traditional medicines. Allosteric mechanisms are also widely involved in the action of SNPs and latent cancer drivers, and can be used in fine and specific tuning of biologics, providing a great potential in diagnostics and therapy. We discuss here major targets for prospected allosteric medicines, currently available allosteric compounds, and drug-candidates at different stages of research and (pre)clinical trials. We describe our computational model of the comprehensive allosteric control of protein activity, outlining the ways of implementing it in pharmacological applications. Finally, we formulate outstanding questions and discuss feasible directions in the work on allosteric drugs and mutations.

Disorder driven allosteric control of protein activity.

Studies of protein allostery increasingly reveal an involvement of the back and forth order-disorder transitions in this mechanism of protein activity regulation. Here, we investigate the allosteric mechanisms mediated by structural disorder using the structure-based statistical mechanical model of allostery (SBSMMA) that we have previously developed. We show that SBSMMA accounts for the energetics and causality of allosteric communication underlying dimerization of the BirA biotin repressor, activation of the sortase A enzyme, and inhibition of the Rac1 GTPase. Using the SBSMMA, we also show that introducing structural order or disorder in various regions of esterases can originate tunable allosteric modulation of the catalytic triad. On the basis of obtained results, we propose that operating with the order-disorder continuum allows one to establish an allosteric control scale for achieving desired modulation of the protein activity.

On the Allosteric Effect of nsSNPs and the Emerging Importance of Allosteric Polymorphism.

The molecular mechanisms of pathological non-synonymous single-nucleotide polymorphisms are still the object of intensive research. To this end, we explore here whether non-synonymous single-nucleotide polymorphisms can work via allosteric mechanisms. Using structure-based statistical mechanical model of allostery and analyzing energetics of the effects of mutations in a set of 27 proteins with at least 50 pathological SNPs in each molecule, we found that, indeed, some SNPs can work allosterically. We illustrate the molecular basis of disease phenotypes caused by allosteric SNPs with the case studies of human galactose 1-phosphate uridyltransferase (GALT) and glucose-6-phosphate dehydrogenase (G6PD). We also found that mutations of a number of other residues in the protein may cause modulation comparable to those observed for known pathological SNPs. In order to explain this, we propose a notion of allosteric polymorphism, which implies the presence of a number of critical positions in the protein sequence, whose mutations can allosterically disrupt the protein function and result in a disease phenotype. We conclude that the emerging importance of allosteric polymorphism calls for the development of computational framework for analyzing the allosteric effects of mutations and their role in the modulation of protein activity.

Towards descriptor of elementary functions for protein design.

We review studies of the protein evolution that help to formulate rules for protein design. Acknowledging the fundamental importance of Dayhoff's provision on the emergence of functional proteins from short peptides, we discuss multiple evidences of the omnipresent partitioning of protein globules into structural/functional units, using which greatly facilitates the engineering and design efforts. Closed loops and elementary functional loops, which are descendants of ancient ring-like peptides that formed fist protein domains in agreement with Dayhoff's hypothesis, can be considered as basic units of protein structure and function. We argue that future developments in protein design approaches should consider descriptors of the elementary functions, which will help to complement designed scaffolds with functional signatures and flexibility necessary for their functions.