Deciphering S100B Allosteric Signaling: The Role of a Peptide Target, TRTK-12, as an Ensemble Modulator.

Allostery is an essential biological phenomenon in which perturbation at one site in a biomolecule elicits a functional response at a distal location(s). It is integral to biological processes, such as cellular signaling, metabolism, and transcription regulation. Understanding allostery is also crucial for rational drug discovery. In this work, we focus on an allosteric S100B protein that belongs to the S100 class of EF-hand Ca2+-binding proteins. The Ca2+-binding affinity of S100B is modulated allosterically by TRTK-12 peptide binding 25 Å away from the Ca2+-binding site. We investigated S100B allostery by carrying out nuclear magnetic resonance (NMR) measurements along with microsecond-long molecular dynamics (MD) simulations on S100B/Ca2+ with/without TRTK-12 at different NaCl salt concentrations. NMR HSQC results show that TRTK-12 reorganizes how S100B/Ca2+ responds to different salt concentrations at both orthosteric and allosteric sites. The MD data suggest that TRTK-12 breaks the dynamic aromatic and hydrogen-bond interactions (not observed in X-ray crystallographic structures) between the hinge/helix and Ca2+-binding EF-hand loop of the two subunits in the homodimeric protein. This triggers rearrangement in the protein network architectures and leads to allosteric communication. Finally, computational studies of S100B at distinct ionic strengths suggest that ligand-bound species are more robust to the changing environment relative to the S100B/Ca2+ complex.

Emergence of allostery through reorganization of protein residue network architecture.

Despite more than a century of study, consensus on the molecular basis of allostery remains elusive. A comparison of allosteric and non-allosteric members of a protein family can shed light on this important regulatory mechanism, and the bacterial biotin protein ligases, which catalyze post-translational biotin addition, provide an ideal system for such comparison. While the Class I bacterial ligases only function as enzymes, the bifunctional Class II ligases use the same structural architecture for an additional transcription repression function. This additional function depends on allosterically activated homodimerization followed by DNA binding. In this work, we used experimental, computational network, and bioinformatics analyses to uncover distinguishing features that enable allostery in the Class II biotin protein ligases. Experimental studies of the Class II Escherichia coli protein indicate that catalytic site residues are critical for both catalysis and allostery. However, allostery also depends on amino acids that are more broadly distributed throughout the protein structure. Energy-based community network analysis of representative Class I and Class II proteins reveals distinct residue community architectures, interactions among the communities, and responses of the network to allosteric effector binding. Bioinformatics mutual information analyses of multiple sequence alignments indicate distinct networks of coevolving residues in the two protein families. The results support the role of divergent local residue community network structures both inside and outside of the conserved enzyme active site combined with distinct inter-community interactions as keys to the emergence of allostery in the Class II biotin protein ligases.

Binding and Functional Folding (BFF): A Physiological Framework for Studying Biomolecular Interactions and Allostery.

EF-hand Ca2+-binding proteins (CBPs), such as S100 proteins (S100s) and calmodulin (CaM), are signaling proteins that undergo conformational changes upon increasing intracellular Ca2+. Upon binding Ca2+, S100 proteins and CaM interact with protein targets and induce important biological responses. The Ca2+-binding affinity of CaM and most S100s in the absence of target is weak (CaKD > 1 µM). However, upon effector protein binding, the Ca2+ affinity of these proteins increases via heterotropic allostery (CaKD < 1 µM). Because of the high number and micromolar concentrations of EF-hand CBPs in a cell, at any given time, allostery is required physiologically, allowing for (i) proper Ca2+ homeostasis and (ii) strict maintenance of Ca2+-signaling within a narrow dynamic range of free Ca2+ ion concentrations, [Ca2+]free. In this review, mechanisms of allostery are coalesced into an empirical "binding and functional folding (BFF)" physiological framework. At the molecular level, folding (F), binding and folding (BF), and BFF events include all atoms in the biomolecular complex under study. The BFF framework is introduced with two straightforward BFF types for proteins (type 1, concerted; type 2, stepwise) and considers how homologous and nonhomologous amino acid residues of CBPs and their effector protein(s) evolved to provide allosteric tightening of Ca2+ and simultaneously determine how specific and relatively promiscuous CBP-target complexes form as both are needed for proper cellular function.

Tuning Allostery through Integration of Disorder to Order with a Residue Network.

In allostery, a signal from one site in a protein is transmitted to a second site to alter its function. Due to its ubiquity in biology and the potential for its exploitation in drug and protein design, the molecular basis of allosteric communication continues to be the subject of intense research. Although allosterically coupled sites are frequently characterized by disorder, how communication between disordered segments occurs remains obscure. Allosteric activation of Escherichia coli BirA dimerization occurs via coupled distant disorder-to-order transitions. In this work, combined structural and computational studies reveal an extensive residue network in BirA. Substitution of several network residues yields large perturbations to allostery. Force distribution analysis reveals that disruptions to the disorder-to-order transitions through amino acid substitution are manifested in shifts in the energy experienced by network residues as well as alterations in packing of an α-helix that plays a critical role in allostery. The combined results reveal a highly distributed allosteric mechanism that is robust to sequence change.