The ensemble nature of allostery.

Allostery describes the ability of biological macromolecules to transmit signals spatially through the molecule from an allosteric site – a site that is distinct from orthosteric binding sites of primary, endogenous ligands – to the functional or active site. This review starts with a historical overview and a description of the classical example of allostery – hemoglobin – and other well-known examples (aspartate transcarbamoylase, Lac repressor, kinases, G-protein-coupled receptors, adenosine triphosphate synthase, and chaperonin). We then discuss fringe examples of allostery, including intrinsically disordered proteins and inter-enzyme allostery, and the influence of dynamics, entropy, and conformational ensembles and landscapes on allosteric mechanisms, to capture the essence of the field. Thereafter, we give an overview over central methods for investigating molecular mechanisms, covering experimental techniques as well as simulations and artificial intelligence (AI)-based methods. We conclude with a review of allostery-based drug discovery, with its challenges and opportunities: with the recent advent of AI-based methods, allosteric compounds are set to revolutionize drug discovery and medical treatments.

Structure and Dynamics of the ATP-Bound Open Conformation of Hsp70 Chaperones

Allosteric regulation refers to the process where the effect of binding of a ligand at one site of a protein is transmitted to another, often distant, functional site. In recent years, it has been demonstrated that allosteric mechanisms can be understood by the conformational ensembles of a protein. Molecular dynamics (MD) simulations are often used for the study of protein allostery as they provide an atomistic view of the dynamics of a protein. However, given the wealth of detailed information hidden in MD data, one has to apply a method that allows extraction of the conformational ensembles underlying allosteric regulation from these data. Markov state models are one of the most promising methods for this purpose. We provide a short introduction to the theory of Markov state models and review their application to various examples of protein allostery studied by MD simulations. We also include a discussion of studies where Markov modelling has been employed to analyse experimental data on allosteric regulation. We conclude our review by advertising the wider application of Markov state models to elucidate allosteric mechanisms, especially since in recent years it has become straightforward to construct such models thanks to software programs like PyEMMA and MSMBuilder.This article is part of a discussion meeting issue 'Allostery and molecular machines'.

Nucleic Acid Conformation Ensembles Revealed by Au-SAXS Interferometry

Metabotropic glutamate receptor 1 (mGlu1), which belongs to class C G protein-coupled receptors (GPCRs), can be coupled with G protein to transfer extracellular signal by dimerization and allosteric regulation. Unraveling the dimer packing and allosteric mechanism can be of great help for understanding specific regulatory mechanism and designing more potential negative allosteric modulator (NAM). Here, we report molecular dynamics simulation studies of the modulation mechanism of FITM on the wild type, T815M and Y805A mutants of mGlu1 through weak interaction analysis and free energy calculation. The weak interaction analysis demonstrates that van der Waals (vdW) and hydrogen bonding play an important role on the dimer packing between six cholesterol molecules and mGlu1 as well as the interaction between allosteric sites T815, Y805 and FITM in wild type, T815M and Y805A mutants of mGlu1. Besides, the results of free energy calculations indicate that secondary binding pocket is mainly formed by the residues Thr748, Cys746, Lys811 and Ser735 except for FITM-bound pocket in crystal structure. Our results can not only reveal the dimer packing and allosteric regulation mechanism, but also can supply useful information for the design of potential NAM of mGlu1.

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) regulates transcription in response to ligand binding at an orthosteric pocket within the ligand-binding domain (LBD). We previously showed that two covalent ligands, T0070907 and GW9662—extensively used as PPARγ inhibitors to assess off-target activity—weaken but do not completely block ligand binding via an allosteric mechanism associated with pharmacological inverse agonism (Shang et al., 2024). These covalent inhibitors shift the LBD towards a repressive conformation, where the activation function-2 (AF-2) helix 12 occupies the orthosteric pocket, competing with orthosteric ligand binding. Here, we provide additional support for this allosteric mechanism using two covalent inverse agonists, SR33065 and SR36708, which better stabilize the repressive LBD conformation and are more effective inhibitors of—but also do not completely inhibit—ligand cobinding. Furthermore, we show that ligand cobinding can occur with a previously reported PPARγ dual-site covalent inhibitor, SR16832, which appears to weaken ligand binding through a direct mechanism independent of the allosteric mechanism. These findings underscore the complex nature of the PPARγ LBD conformational ensemble and highlight the need to develop alternative methods for designing more effective covalent inhibitors.

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) regulates transcription in response to ligand binding at an orthosteric pocket within the ligand-binding domain (LBD). We previously showed that two covalent ligands, T0070907 and GW9662-extensively used as PPARγ inhibitors to assess off-target activity-weaken but do not completely block ligand binding via an allosteric mechanism associated with pharmacological inverse agonism (Shang and Kojetin, 2024). These covalent inhibitors shift the LBD toward a repressive conformation, where the activation function-2 (AF-2) helix 12 occupies the orthosteric pocket, competing with orthosteric ligand binding. Here, we provide additional support for this allosteric mechanism using two covalent inverse agonists, SR33065 and SR36708, which better stabilize the repressive LBD conformation and are more effective inhibitors of-but also do not completely inhibit-ligand cobinding. Furthermore, we show that ligand cobinding can occur with a previously reported PPARγ dual-site covalent inhibitor, SR16832, which appears to weaken ligand binding through a direct mechanism independent of the allosteric mechanism. These findings underscore the complex nature of the PPARγ LBD conformational ensemble and highlight the need to develop alternative methods for designing more effective covalent inhibitors.

Proteins are dynamic entities that populate conformational ensembles, and most functions of proteins depend on their dynamic character. Allostery, in particular, relies on ligand-modulated shifts in these conformational ensembles. Hsp70s are allosteric molecular chaperones with conformational landscapes that involve large rearrangements of their two domains (viz. the nucleotide-binding domain and substrate-binding domain) in response to adenine nucleotides and substrates. However, it remains unclear how the Hsp70 conformational ensemble is populated at each point of the allosteric cycle and how ligands control these populations. We have mapped the conformational species present under different ligand-binding conditions throughout the allosteric cycle of the Escherichia coli Hsp70 DnaK by two complementary methods, ion-mobility mass spectrometry and double electron-electron resonance. Our results obtained under biologically relevant ligand-bound conditions confirm the current picture derived from NMR and crystallographic data of domain docking upon ATP binding and undocking in response to ADP and substrate. Additionally, we find that the helical lid of DnaK is a highly dynamic unit of the structure in all ligand-bound states. Importantly, we demonstrate that DnaK populates a partially docked state in the presence of ATP and substrate and that this state represents an energy minimum on the DnaK allosteric landscape. Because Hsp70s are emerging as potential drug targets for many diseases, fully mapping an allosteric landscape of a molecular chaperone like DnaK will facilitate the development of small molecules that modulate Hsp70 function via allosteric mechanisms.

Prediction of Allosteric Linkages in Proteins

Parvulins are a group of peptidyl-prolyl isomerases (PPIases) responsible for important biological processes in all kingdoms of life. The PinA protein from the psychrophilic archaeon Cenarchaeum symbiosum is a parvulin-like PPIase. Due to its striking similarity to the human parvulins Pin1 and Par14, PinA constitutes an interesting subject for structural and functional studies. Here, we present the first high resolution NMR structure of an archaeal parvulin, PinA, based on 1798 conformational restraints. Structure calculation yields an ensemble of 20 convergent low energy structures with a backbone r.m.s.d. value of 0.6 Å within the secondary structure elements. The overall fold of PinA comprises the β-α3-β-α-β2 fold typical for all parvulin structures known so far, but with helix III being a short 310-helix. A detailed comparison of this high resolution structure of the first archaeal PinA protein with bacterial and eukaryotic parvulin PPIase structures reveals an atypically large catalytic binding site. This feature provides an explanation for cold-adapted protein function. Moreover, the residues in and around 310-helix III exhibit strong intramolecular dynamics on a microsecond to millisecond timescale and display structural heterogeneity within the NMR ensemble. A putative peptide ligand was found for PinA by phage display and was used for 1H-15N-HSQC titrations. Again, the flexible region around 310-helix III as well as residues of the peptide binding pocket showed the strongest chemical shift perturbations upon peptide binding. The local flexibility of this region also was modulated by ligand binding. A glycine and two positively charged residues are conserved in most parvulin proteins in this flexible loop region, which may be of general functional importance for parvulin-type PPIases.

Nicotinamide adenine dinucleotide synthetases (NADS) catalyze the amidation of nicotinic acid adenine dinucleotide (NAAD) to yield the enzyme cofactor nicotinamide adenine dinucleotide (NAD). Here we describe the crystal structures of the ammonia-dependent homodimeric NADS from Escherichia coli alone and in complex with natural substrates and with the reaction product NAD. The structures disclosed two NAAD/NAD binding sites at the dimer interface and an adenosine triphosphate (ATP) binding site within each subunit. Comparison with the Bacillus subtilis NADS showed pronounced chemical differences in the NAAD/NAD binding sites and less prominent differences in the ATP binding pockets. In addition, the E. coli NADS structures revealed unexpected dynamical rearrangements in the NAAD/NAD binding pocket upon NAAD-to-NAD conversion, which define a catalysis state and a substrate/product exchange state. The two states are adopted by concerted movement of the nicotinysyl moieties of NAAD and NAD, Phe-170, and residues 224-228, which may be triggered by differential coordination of a magnesium ion to NAAD and NAD. Phylogenetic structure comparisons suggest that the present results are relevant for designing species-specific antibiotics.

Combining Biophysical Experiments and Biomolecular Simulations

Multidomain proteins containing both folded and intrinsically disordered regions are crucial for biological processes, but characterizing their conformational ensembles and dynamics remains challenging. We introduce the Quality Evaluation Based Simulation Selection (QEBSS) protocol, which combines MD simulations with NMR-derived protein backbone 15N T1 and T2 spin relaxation times and hetNOE values to interpret conformational ensembles and dynamics of multidomain proteins. We demonstrate the practical advantage of QEBSS by characterizing four flexible multidomain proteins: calmodulin, EN2, MANF, and CDNF. These biologically important proteins have been difficult to study due to their flexible nature. Our findings reveal new insights into their conformational landscapes and dynamics, providing mechanistic understanding of their biological functions. QEBSS offers quantitative quality evaluation of simulations and a systematic approach for resolving conformational ensembles of multidomain proteins with heterogeneous dynamics. Given the importance of such proteins in biology, biotechnology, and materials science, QEBSS should benefit fields from drug design to novel materials development.

Functional and computational studies reveal that the allosteric inhibition mechanism is conserved between glutamate and neutral amino acid transporters of the SLC1 family, and identify a novel allosteric inhibitor.

Functional and computational studies reveal that the allosteric inhibition mechanism is conserved between glutamate and neutral amino acid transporters of the SLC1 family, and identify a novel allosteric inhibitor.