Unveiling the Complexity of cis-Regulation Mechanisms in Kinases: A Comprehensive Analysis.

Protein cis-regulatory elements (CREs) are regions that modulate the activity of a protein through intramolecular interactions. Kinases, pivotal enzymes in numerous biological processes, often undergo regulatory control via inhibitory interactions in cis. This study delves into the mechanisms of cis regulation in kinases mediated by CREs, employing a combined structural and sequence analysis. To accomplish this, we curated an extensive dataset of kinases featuring annotated CREs, organized into homolog families through multiple sequence alignments. Key molecular attributes, including disorder and secondary structure content, active and ATP-binding sites, post-translational modifications, and disease-associated mutations, were systematically mapped onto all sequences. Additionally, we explored the potential for conformational changes between active and inactive states. Finally, we explored the presence of these kinases within membraneless organelles and elucidated their functional roles therein. CREs display a continuum of structures, ranging from short disordered stretches to fully folded domains. The adaptability demonstrated by CREs in achieving the common goal of kinase inhibition spans from direct autoinhibitory interaction with the active site within the kinase domain, to CREs binding to an alternative site, inducing allosteric regulation revealing distinct types of inhibitory mechanisms, which we exemplify by archetypical representative systems. While this study provides a systematic approach to comprehend kinase CREs, further experimental investigations are imperative to unravel the complexity within distinct kinase families. The insights gleaned from this research lay the foundation for future studies aiming to decipher the molecular basis of kinase dysregulation, and explore potential therapeutic interventions.

Substrate Specificity of GSDA Revealed by Cocrystal Structures and Binding Studies.

In plants, guanosine deaminase (GSDA) catalyzes the deamination of guanosine for nitrogen recycling and re-utilization. We previously solved crystal structures of GSDA from Arabidopsis thaliana (AtGSDA) and identified several novel substrates for this enzyme, but the structural basis of the enzyme activation/inhibition is poorly understood. Here, we continued to solve 8 medium-to-high resolution (1.85-2.60 Å) cocrystal structures, which involved AtGSDA and its variants bound by a few ligands, and investigated their binding modes through structural studies and thermal shift analysis. Besides the lack of a 2-amino group of these guanosine derivatives, we discovered that AtGSDA's inactivity was due to the its inability to seclude its active site. Furthermore, the C-termini of the enzyme displayed conformational diversities under certain circumstances. The lack of functional amino groups or poor interactions/geometries of the ligands at the active sites to meet the precise binding and activation requirements for deamination both contributed to AtGSDA's inactivity toward the ligands. Altogether, our combined structural and biochemical studies provide insight into GSDA.

Molecular crowding accelerates aggregation of α-synuclein by altering its folding pathway.

Intracellular macromolecular crowding can lead to increased aggregation of proteins, especially those that lack a natively folded conformation. Crowding may also be mimicked by the addition of polymers like polyethylene glycol (PEG) in vitro. α-Synuclein is an intrinsically disordered protein that exhibits increased aggregation and amyloid fibril formation in a crowded environment. Two hypotheses have been proposed to explain this observation. One is the excluded volume effect positing that reduced water activity in a crowded environment leads to increased effective protein concentration, promoting aggregation. An alternate explanation is that increased crowding facilitates conversion to a non-native form increasing the rate of aggregation. In this work, we have segregated these two hypotheses to investigate which one is operating. We show that mere increase in concentration of α-synuclein is not enough to induce aggregation and consequent fibrillation. In vitro, we find a complex relationship between PEG concentrations and aggregation, in which smaller PEGs delay fibrillation; while, larger ones promote fibril nucleation. In turn, while PEG600 did not increase the rate of aggregation, PEG1000 did and PEG4000 and PEG12000 slowed it but led to a higher overall fibril burden in the latter to cases. In cells, PEG4000 reduces the aggregation of α-synuclein but in a way specific to the cellular environment/due to cellular factors. The aggregation of the similarly sized, globular lysozyme does not increase in vitro when at the same concentrations with either PEG8000 or PEG12000. Thus, natively disordered α-synuclein undergoes a conformational transition in specific types of crowded environment, forming an aggregation-prone conformer.

Extant fold-switching proteins are widespread.

A central tenet of biology is that globular proteins have a unique 3D structure under physiological conditions. Recent work has challenged this notion by demonstrating that some proteins switch folds, a process that involves remodeling of secondary structure in response to a few mutations (evolved fold switchers) or cellular stimuli (extant fold switchers). To date, extant fold switchers have been viewed as rare byproducts of evolution, but their frequency has been neither quantified nor estimated. By systematically and exhaustively searching the Protein Data Bank (PDB), we found ∼100 extant fold-switching proteins. Furthermore, we gathered multiple lines of evidence suggesting that these proteins are widespread in nature. Based on these lines of evidence, we hypothesized that the frequency of extant fold-switching proteins may be underrepresented by the structures in the PDB. Thus, we sought to identify other putative extant fold switchers with only one solved conformation. To do this, we identified two characteristic features of our ∼100 extant fold-switching proteins, incorrect secondary structure predictions and likely independent folding cooperativity, and searched the PDB for other proteins with similar features. Reassuringly, this method identified dozens of other proteins in the literature with indication of a structural change but only one solved conformation in the PDB. Thus, we used it to estimate that 0.5-4% of PDB proteins switch folds. These results demonstrate that extant fold-switching proteins are likely more common than the PDB reflects, which has implications for cell biology, genomics, and human health.

Simple yet functional phosphate-loop proteins.

Abundant and essential motifs, such as phosphate-binding loops (P-loops), are presumed to be the seeds of modern enzymes. The Walker-A P-loop is absolutely essential in modern NTPase enzymes, in mediating binding, and transfer of the terminal phosphate groups of NTPs. However, NTPase function depends on many additional active-site residues placed throughout the protein's scaffold. Can motifs such as P-loops confer function in a simpler context? We applied a phylogenetic analysis that yielded a sequence logo of the putative ancestral Walker-A P-loop element: a ß-strand connected to an α-helix via the P-loop. Computational design incorporated this element into de novo designed ß-α repeat proteins with relatively few sequence modifications. We obtained soluble, stable proteins that unlike modern P-loop NTPases bound ATP in a magnesium-independent manner. Foremost, these simple P-loop proteins avidly bound polynucleotides, RNA, and single-strand DNA, and mutations in the P-loop's key residues abolished binding. Binding appears to be facilitated by the structural plasticity of these proteins, including quaternary structure polymorphism that promotes a combined action of multiple P-loops. Accordingly, oligomerization enabled a 55-aa protein carrying a single P-loop to confer avid polynucleotide binding. Overall, our results show that the P-loop Walker-A motif can be implemented in small and simple ß-α repeat proteins, primarily as a polynucleotide binding motif.

Design and Synthesis of 56 Shape-Diverse 3D Fragments.

Fragment-based drug discovery is now widely adopted for lead generation in the pharmaceutical industry. However, fragment screening collections are often predominantly populated with flat, 2D molecules. Herein, we describe a workflow for the design and synthesis of 56 3D disubstituted pyrrolidine and piperidine fragments that occupy under-represented areas of fragment space (as demonstrated by a principal moments of inertia (PMI) analysis). A key, and unique, underpinning design feature of this fragment collection is that assessment of fragment shape and conformational diversity (by considering conformations up to 1.5 kcal mol-1 above the energy of the global minimum energy conformer) is carried out prior to synthesis and is also used to select targets for synthesis. The 3D fragments were designed to contain suitable synthetic handles for future fragment elaboration. Finally, by comparing our 3D fragments with six commercial libraries, it is clear that our collection has high three-dimensionality and shape diversity.

Drug Discovery Targeting the Disorder-To-Order Transition Regions through the Conformational Diversity Mimicking and Statistical Analysis.

Intrinsically disordered proteins exist as highly dynamic conformational ensembles of diverse forms. However, the majority of virtual screening only focuses on proteins with defined structures. This means that computer-aided drug discovery is restricted. As a breakthrough, understanding the structural characteristics of intrinsically disordered proteins and its application can open the gate for unrestricted drug discovery. First, we segmented the target disorder-to-order transition region into a series of overlapping 20-amino-acid-long peptides. Folding prediction generated diverse conformations of these peptides. Next, we applied molecular docking, new evaluation score function, and statistical analysis. This approach successfully distinguished known compounds and their corresponding binding regions. Especially, Myc proto-oncogene protein (MYC) inhibitor 10058F4 was well distinguished from others of the chemical compound library. We also studied differences between the two Methyl-CpG-binding domain protein 2 (MBD2) inhibitors (ABA (2-amino-N-[[(3S)-2,3-dihydro-1,4-benzodioxin-3-yl]methyl]-acetamide) and APC ((R)-(3-(2-Amino-acetylamino)-pyrrolidine-1-carboxylic acid tert-butyl ester))). Both compounds bind MBD2 through electrostatic interaction behind its p66α-binding site. ABA is also able to bind p66α through electrostatic interaction behind its MBD2-binding site while APC-p66α binding was nonspecific. Therefore, structural heterogeneity mimicking of the disorder-to-order transition region at the peptide level and utilization of the new docking score function represent a useful approach that can efficiently discriminate compounds for expanded virtual screening toward intrinsically disordered proteins.

Evolutionary rate heterogeneity between multi- and single-interface hubs across human housekeeping and tissue-specific protein interaction network: Insights from proteins' and its partners' properties.

Integrating gene expression into protein-protein interaction network (PPIN) leads to the construction of tissue-specific (TS) and housekeeping (HK) sub-networks, with distinctive TS- and HK-hubs. All such hub proteins are divided into multi-interface (MI) hubs and single-interface (SI) hubs, where MI hubs evolve slower than SI hubs. Here we explored the evolutionary rate difference between MI and SI proteins within TS- and HK-PPIN and observed that this difference is present only in TS, but not in HK-class. Next, we explored whether proteins' own properties or its partners' properties are more influential in such evolutionary discrepancy. Statistical analyses revealed that this evolutionary rate correlates negatively with protein's own properties like expression level, miRNA count, conformational diversity and functional properties and with its partners' properties like protein disorder and tissue expression similarity. Moreover, partial correlation and regression analysis revealed that both proteins' and its partners' properties have independent effects on protein evolutionary rate.

Large scale analysis of protein conformational transitions from aqueous to non-aqueous media.

BACKGROUND: Biocatalysis in organic solvents is nowadays a common practice with a large potential in Biotechnology. Several studies report that proteins which are co-crystallized or soaked in organic solvents preserve their fold integrity showing almost identical arrangements when compared to their aqueous forms. However, it is well established that the catalytic activity of proteins in organic solvents is much lower than in water. In order to explain this diminished activity and to further characterize the behaviour of proteins in non-aqueous environments, we performed a large-scale analysis (1737 proteins) of the conformational diversity of proteins crystallized in aqueous and co-crystallized or soaked in non-aqueous media. RESULTS: Using proteins' experimentally determined conformational diversity taken from CoDNaS database, we found that proteins in non-aqueous media display much lower conformational diversity when compared to the corresponding conformers obtained in water. When conformational diversity is compared between conformers obtained in different non-aqueous media, their structural differences are larger and mostly independent of the presence of cognate ligands. We also found that conformers corresponding to non-aqueous media have larger but less flexible cavities, lower number of disordered regions and lower active-site residue mobility. CONCLUSIONS: Our results show that non-aqueous media conformers have specific structural features and that they do not adopt extreme conformations found in aqueous media. This makes them clearly different from their corresponding aqueous conformers.

How is structural divergence related to evolutionary information?

The analysis of evolutionary information in a protein family, such as conservation and covariation, is often linked to its structural information. Multiple sequence alignments of distant homologous sequences are used to measure evolutionary variables. Although high structural differences between proteins can be expected in such divergent alignments, most works linking evolutionary and structural information use a single structure ignoring the structural variability within protein families. The goal of this work is to elucidate the relevance of structural divergence when sequence-based measures are integrated with structural information. We found that inter-residue contacts and solvent accessibility undergo large variations in protein families. Our results show that high covariation scores tend to reveal residue contacts that are conserved in the family, instead of protein or conformer specific contacts. We also found that residue accessible surface area shows a high variability between structures of the same family. As a consequence, the mean relative solvent accessibility of multiple structures correlates better with the conservation pattern than the relative solvent accessibility of a single structure. We conclude that the use of comprehensive structural information allows a more accurate interpretation of the information computed from sequence alignments. Therefore, considering structural divergence would lead to a better understanding of protein function, dynamics, and evolution.

Protein conformational diversity correlates with evolutionary rate.

Native state of proteins is better represented by an ensemble of conformers in equilibrium than by only one structure. The extension of structural differences between conformers characterizes the conformational diversity of the protein. In this study, we found a negative correlation between conformational diversity and protein evolutionary rate. Conformational diversity was expressed as the maximum root mean square deviation (RMSD) between the available conformers in Conformational Diversity of Native State database. Evolutionary rate estimations were calculated using 16 different species compared with human sharing at least 700 orthologous proteins with known conformational diversity extension. The negative correlation found is independent of the protein expression level and comparable in magnitude and sign with the correlation between gene expression level and evolutionary rate. Our findings suggest that the structural constraints underlying protein dynamism, essential for protein function, could modulate protein divergence.

Combining Protein Conformational Diversity and Phylogenetic Information Using CoDNaS and CoDNaS-Q.

CoDNaS (http://ufq.unq.edu.ar/codnas/) and CoDNaS-Q (http://ufq.unq.edu.ar/codnasq) are repositories of proteins with different degrees of conformational diversity. Following the ensemble nature of the native state, conformational diversity represents the structural differences between the conformers in the ensemble. Each entry in CoDNaS and CoDNaS-Q contains a redundant collection of experimentally determined conformers obtained under different conditions. These conformers represent snapshots of the protein dynamism. While CoDNaS contains examples of conformational diversity at the tertiary level, a recent development, CoDNaS-Q, contains examples at the quaternary level. In the emerging age of accurate protein structure prediction by machine learning approaches, many questions remain open regarding the characterization of protein dynamism. In this context, most bioinformatics resources take advantage of distinct features derived from protein alignments, however, the complexity and heterogeneity of information makes it difficult to recover reliable biological signatures. Here we present five protocols to explore tertiary and quaternary conformational diversity at the individual protein level as well as for the characterization of the distribution of conformational diversity at the protein family level in a phylogenetic context. These protocols can provide curated protein families with experimentally known conformational diversity, facilitating the exploration of sequence determinants of protein dynamism. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Assessing conformational diversity with CoDNaS Alternate Protocol 1: Assessing conformational diversity at the quaternary level with CoDNaS-Q Basic Protocol 2: Exploring conformational diversity in a protein family Alternate Protocol 2: Exploring quaternary conformational diversity in a protein family Basic Protocol 3: Representing conformational diversity in a phylogenetic context.

Easy Not Easy: Comparative Modeling with High-Sequence Identity Templates.

Homology modeling is the most common technique to build structural models of a target protein based on the structure of proteins with high-sequence identity and available high-resolution structures. This technique is based on the idea that protein structure shows fewer changes than sequence through evolution. While in this scenario single mutations would minimally perturb the structure, experimental evidence shows otherwise: proteins with high conformational diversity impose a limit of the paradigm of comparative modeling as the same protein sequence can adopt dissimilar three-dimensional structures. These cases present challenges for modeling; at first glance, they may seem to be easy cases, but they have a complexity that is not evident at the sequence level. In this chapter, we address the following questions: Why should we care about conformational diversity? How to consider conformational diversity when doing template-based modeling in a practical way?

Evolving prion-like tau conformers differentially alter postsynaptic proteins in neurons inoculated with distinct isolates of Alzheimer's disease tau.

OBJECTIVES: Although accumulation of misfolded tau species has been shown to predict cognitive decline in patients with Alzheimer's disease (AD) and other tauopathies but with the remarkable diversity of clinical manifestations, neuropathology profiles, and time courses of disease progression remaining unexplained by current genetic data. We considered the diversity of misfolded tau conformers present in individual AD cases as an underlying driver of the phenotypic variations of AD and progressive loss of synapses. METHODS: To model the mechanism of tau propagation and synaptic toxicity of distinct tau conformers, we inoculated wild-type primary mouse neurons with structurally characterized Sarkosyl-insoluble tau isolates from the frontal cortex of six AD cases and monitored the impact for fourteen days. We analyzed the accumulation rate, tau isoform ratio, and conformational characteristics of de novo-induced tau aggregates with conformationally sensitive immunoassays, and the dynamics of synapse formation, maintenance, and their loss using a panel of pre-and post-synaptic markers. RESULTS: At the same concentrations of tau, the different AD tau isolates induced accumulation of misfolded predominantly 4-repeat tau aggregates at different rates in mature neurons, and demonstrated distinct conformational characteristics corresponding to the original AD brain tau. The time-course of the formation of misfolded tau aggregates and colocalization correlated with significant loss of synapses in tau-inoculated cell cultures and the reduction of synaptic connections implicated the disruption of postsynaptic compartment as an early event. CONCLUSIONS: The data obtained with mature neurons expressing physiological levels and adult isoforms of tau protein demonstrate markedly different time courses of endogenous tau misfolding and differential patterns of post-synaptic alterations. These and previous biophysical data argue for an ensemble of various misfolded tau aggregates in individual AD brains and template propagation of their homologous conformations in neurons with different rates and primarily postsynaptic interactors. Modeling tau aggregation in mature differentiated neurons provides a platform for investigating divergent molecular mechanisms of tau strain propagation and for identifying common structural features of misfolded tau and critical interactors for new therapeutic targets and approaches in AD.

Rational design of PIN1 inhibitors for cancer treatment based on conformational diversity analysis and docking based virtual screening.

The parvulin PIN1 (peptidyl-prolyl cis-trans isomerase NIMA-interacting 1), is the only enzyme capable of isomerizing prolines of phospho-Serine/Threonine-Proline motifs. PIN1 binds to a subset of proteins and plays an essential role in regulating protein function post-phosphorylation control. Furthermore, the activity of PIN1 regulates the outcome of the signalling of proline-directed kinases (e.g. MAPK, CDK, or GSK3) and thus regulates cell proliferation and cell survival. For these reasons, PIN1 inhibitors are interesting since they may have therapeutic implications for cancer. Several authors have already reported that the non-structural point mutation Trp34Ala prevents PIN1 from interacting with its downstream effector proteins. In this work, we characterized PIN1 structurally, intending to explore new inhibition targets for the rational design of pharmacological activity compounds. Through a conformational diversity analysis of PIN1, we identified and characterized a highly specific druggable pocket around the residue Trp34. This pocket was used in a high-throughput docking screening of 450,000 drug-like compounds, and the top 10 were selected for re-docking studies on the previously used conformers. Finally, we evaluated the binding of each compound by thermal shift assay and found four molecules with a high affinity for PIN1 and potential inhibitory activity. Through this strategy, we achieved novel drug candidates with the ability to interfere with the phosphorylation-dependent actions of PIN1 and with potential applications in the treatment of cancer.Communicated by Ramaswamy H. Sarma.

Design and Synthesis of Conformationally Diverse Pyrimidine-Embedded Medium/Macro- and Bridged Cycles via Skeletal Transformation.

The rigidity and flexibility of small molecules are complementary in 3-dimensional ligand-protein interaction. Therefore, the chemical library with conformational diversity would be a valuable resource for investigating the influence of skeletal flexibility on the biological system. In this regard, we designed and synthesized ten conformationally diverse pyrimidine-embedded medium/macro- and bridged cyclic scaffolds covering 7- to 14-member rings via an efficient skeletal transformation strategy. Their high conformational and shape diversity was confirmed by chemoinformatic analysis.

Case for an RNA-prion world: a hypothesis based on conformational diversity.

Prions and other misfolded proteins can impart their structure and functions to normal molecules. Based upon a thorough structural assessment of RNA, prions and misfolded proteins, especially from the perspective of conformational diversity, we propose a case for co-existence of these in the pre-biotic world. Analyzing the evolution of physical aspects of biochemical structures, we put forward a case for an RNA-prion pre-biotic world, instead of, merely, the "RNA World".

Intrinsically Disordered Protein Ensembles Shape Evolutionary Rates Revealing Conformational Patterns.

Intrinsically disordered proteins (IDPs) lack stable tertiary structure under physiological conditions. The unique composition and complex dynamical behaviour of IDPs make them a challenge for structural biology and molecular evolution studies. Using NMR ensembles, we found that IDPs evolve under a strong site-specific evolutionary rate heterogeneity, mainly originated by different constraints derived from their inter-residue contacts. Evolutionary rate profiles correlate with the experimentally observed conformational diversity of the protein, allowing the description of different conformational patterns possibly related to their structure-function relationships. The correlation between evolutionary rates and contact information improves when structural information is taken not from any individual conformer or the whole ensemble, but from combining a limited number of conformers. Our results suggest that residue contacts in disordered regions constrain evolutionary rates to conserve the dynamic behaviour of the ensemble and that evolutionary rates can be used as a proxy for the conformational diversity of IDPs.

Chiral Conformation of Subnanometric Materials.

Subnanometric materials (SNMs) refer to nanomaterials with sizes comparable to the diameter of common linear polymers or confined at the level of a single unit cell in at least one dimension, usually <1 nm. Conventional inorganic nanoparticles are usually deemed to be rigid, lacking self-adjustable conformation. In contrast, the size at subnanometric scale endows SNMs with flexibility analogous to polymers, resulting in their abundant self-adjustable conformation. It is noteworthy that some highly flexible SNMs can adjust their shape automatically to form chiral conformation, which is rare in conventional inorganic nanoparticles. Herein, we summarize the chiral conformation of SNMs and clarify the driving force behind their formation, in an attempt to establish a better understanding for the origin of flexibility and chirality at subnanometric scale. In addition, the general strategies for controlling the conformation of SNMs are elaborated, which might shed light on the efficient fabrications of chiral inorganic materials. Finally, the challenges facing this area as well as some unexplored topics are discussed.

Conformational diversity of the quercetin molecule: a quantum-chemical view.

This paper focuses on the comprehensive conformational analysis of the quercetin molecule with a broad range of the therapeutic and biological actions. All possible conformers of these molecule, corresponding to the local minima on the potential energy hypersurface, have been obtained by the sequential rotation of its five hydroxyl groups and also by the rotation of its (A + C) and B rings relatively each other. Altogether, it was established 48 stable conformers, among which 24 conformers possess planar structure and 24 conformers - nonplanar structure. Their structural, symmetrical, energetical and polar characteristics have been investigated in details. Quantum-mechanical calculations indicate that conformers of the quercetin molecule are polar structures with a dipole moment, which varies within the range from 0.35 to 9.87 Debay for different conformers. Relative Gibbs free energies of these conformers are located within the range from 0.0 to 25.3 kcal·mol-1 in vacuum under normal conditions. Impact of the continuum with ε = 4 leads to the decreasing of the Gibbs free energies (-0.19-18.15 kcal·mol-1) and increasing of the dipole moment (0.57-12.48 D). It was shown that conformers of the quercetin molecule differ from each other by the intramolecular specific contacts (two or three), stabilizing all possible conformers of the molecule - H-bonds (both classical ОН…Ðž and so-called unusual С'Н…Ðž and ОН…Ð¡') and attractive van-der-Waals contacts О…Ðž. Obtained conformational analysis for the quercetin molecule enables to provide deeper understanding of the 'structure-function' relationship and also to suggest its mechanisms of the therapeutic and biological actions.Communicated by Ramaswamy H. Sarma.