Allosteric Response Research Articles

Cooperative folding as a molecular switch in an evolved antibody binder

Designing proteins with tunable activities from easily accessible external cues remains a biotechnological challenge. Here, we set out to create a small antibody-binding domain equipped with a molecular switch inspired by the allosteric response to calcium seen in naturally derived proteins like calmodulin. We have focused on one of the three domains of Protein G that show inherent affinity to antibodies. By combining a semi-rational protein design with directed evolution, we engineered novel variants containing a calcium-binding loop rendering the inherent antibody affinity calcium-dependent. The evolved variants resulted from a designed selection strategy subjecting them to negative and positive selection pressures focused on conditional antibody-binding. Hence, these variants contained molecular “on/off” switches, controlling the target affinity towards antibody fragments simply by the presence or absence of calcium. From NMR spectroscopy we found that the molecular mechanism underlying the evolved switching behavior was a coupled calcium-binding and folding event where the target binding surface was intact and functional only in the presence of bound calcium. Notably, it was observed that the response to the employed selection pressures gave rise to the evolution of a cooperative folding mechanism. This observation illustrates why the cooperative folding reaction is an effective solution seen repeatedly in the natural evolution of fine-tuned macromolecular recognition. Engineering binding moieties to confer conditional target interaction has great potential due to the exquisite interaction control that is tunable to application requirements. Improved understanding of the molecular mechanisms behind regulated interactions is crucial to unlock how to engineer switchable proteins useful in a variety of biotechnological applications.

An expanded trove of fragment-bound structures for the allosteric enzyme PTP1B from computational reanalysis of large-scale crystallographic data

Due to their low binding affinities, detecting small-molecule fragments bound to protein structures from crystallographic datasets has been a challenge. Here, we report a trove of 65 new fragment hits for PTP1B, an "undruggable" therapeutic target enzyme for diabetes and cancer. These structures were obtained from computational analysis of data from a large crystallographic screen, demonstrating the power of this approach to elucidate many (∼50% more) "hidden" ligand-bound states of proteins. Our new structures include a fragment hit found in a novel binding site in PTP1B with a unique location relative to the active site, one that links adjacent allosteric sites, and, perhaps most strikingly, a fragment that induces long-range allosteric protein conformational responses. Altogether, our research highlights the utility of computational analysis of crystallographic data, makes publicly available dozens of new ligand-bound structures of a high-value drug target, and identifies novel aspects of ligandability and allostery in PTP1B.

Native dynamics and allosteric responses in PTP1B probed by high-resolution HDX-MS.

Protein tyrosine phosphatase 1B (PTP1B) is a validated therapeutic target for obesity, diabetes, and certain types of cancer. In particular, allosteric inhibitors hold potential for therapeutic use, but an incomplete understanding of conformational dynamics and allostery in this protein has hindered their development. Here, we interrogate solution dynamics and allosteric responses in PTP1B using high-resolution hydrogen-deuterium exchange mass spectrometry (HDX-MS), an emerging and powerful biophysical technique. Using HDX-MS, we obtain a detailed map of backbone amide exchange that serves as a proxy for the solution dynamics of apo PTP1B, revealing several flexible loops interspersed among more constrained and rigid regions within the protein structure, as well as local regions that exchange faster than expected from their secondary structure and solvent accessibility. We demonstrate that our HDX rate data obtained in solution adds value to estimates of conformational heterogeneity derived from a pseudo-ensemble constructed from ~200 crystal structures of PTP1B. Furthermore, we report HDX-MS maps for PTP1B with active-site versus allosteric small-molecule inhibitors. These maps suggest distinct and widespread effects on protein dynamics relative to the apo form, including changes in locations distal (>35 Å) from the respective ligand binding sites. These results illuminate that allosteric inhibitors of PTP1B can induce unexpected changes in dynamics that extend beyond the previously understood allosteric network. Together, our data suggest a model of BB3 allostery in PTP1B that combines conformational restriction of active-site residues with compensatory liberation of distal residues that aid in entropic balancing. Overall, our work showcases the potential of HDX-MS for elucidating aspects of protein conformational dynamics and allosteric effects of small-molecule ligands and highlights the potential of integrating HDX-MS alongside other complementary methods, such as room-temperature X-ray crystallography, NMR spectroscopy, and molecular dynamics simulations, to guide the development of new therapeutics.

Synergistic inhibition mechanism of quinazolinone and piperacillin on penicillin-binding protein 2a: a promising approach for combating methicillin-resistant Staphylococcus aureus

The production of penicillin-binding protein 2a (PBP2a), a cell wall synthesis protein, is primarily responsible for the high-level resistance observed in methicillin-resistant Staphylococcus aureus (MRSA). PBP2a exhibits a significantly reduced affinity for most β-lactam antibiotics owing to its tightly closed active site. Quinazolinones (QNE), a novel class of non-β-lactam antibiotics, could initiate the allosteric regulation of PBP2a, resulting in the opening of the initially closed active pocket. Based on our previous study, we have a basic understanding of the dual-site inhibitor ceftaroline (CFT) induced allosteric regulation of PBP2a. However, there are still limitations in the knowledge of how combining medicines, QNE and piperacillin (PIP), induce the allosteric response of PBP2a and inhibit its function. Herein, molecular dynamics (MD) simulations were performed to elucidate the intricate mechanisms underlying the combination mode of QNE and PIP. Our study successfully captured the opening process of the active pocket upon the binding of the QNE at the allosteric site, which alters the signaling pathways with a favorable transmission to the active site. Subsequent docking experiments with different conformational states of the active pocket indicated that all three inhibitors, PIP, QNE, and CFT, exhibited higher docking scores and more favorable docking poses to the open active pocket. These findings reveal the implied mechanism of QNE-mediated allostery underlying combination therapy and provide novel insights into developing innovative therapeutic modalities against MRSA. Communicated by Ramaswamy H. Sarma

Structural response of G protein binding to the cyclodepsipeptide inhibitor FR900359 probed by NMR spectroscopy.

The cyclodepsipeptide FR900359 (FR) and its analogs are able to selectively inhibit the class of Gq proteins by blocking GDP/GTP exchange. The inhibitor binding site of Gq has been characterized by X-ray crystallography, and various binding and functional studies have determined binding kinetics and mode of inhibition. Here we investigate isotope-labeled FR bound to the membrane-anchored G protein heterotrimer by solid-state nuclear magnetic resonance (ssNMR) and in solution by liquid-state NMR. The resulting data allowed us to identify regions of the inhibitor which show especially pronounced effects upon binding and revealed a generally rigid binding mode in the cis conformation under native-like conditions. The inclusion of the membrane environment allowed us to show a deep penetration of FR into the lipid bilayer illustrating a possible access mode of FR into the cell. Dynamic nuclear polarization (DNP)-enhanced ssNMR was used to observe the structural response of specific segments of the Gα subunit to inhibitor binding. This revealed rigidification of the switch I binding site and an allosteric response in the α5 helix as well as suppression of structural changes induced by nucleotide exchange due to inhibition by FR. Our NMR studies of the FR-G protein complex conducted directly within a native membrane environment provide important insights into the inhibitors access via the lipid membrane, binding mode, and structural allosteric effects.

Partner Gene Associated Trinary Fusion and Orchestration of Multiple Molecular Events in Acute Promyelocytic Leukemia with Variant Rara Fusion Genes

Background All-trans retinoic acid (ATRA) has been used with great success in acute promyelocytic leukemia (APL) cases with PML::RARA fusion gene (FG). There are still PML::RARA negative cases manifest as APL. So far, 19 RARA fusion partner genes have been reported, with all reported as 5′ partners ( X::RARA). Intriguingly, a significant proportion of X::RARA cases were resistant to ATRA, and mechanisms are yet to be elucidated. Here we report the finding of a novel type of trinary fusion and the orchestrating of multiple molecular events that confer leukemogenesis and ATRA resistance in X::RARA-APL. Methods A total of 23 cases (Fig. 1A) with RNA-seq and WGS data from 10 centers were enrolled. We used Arriba for routine FG calling and manually investigated the nonconventional fusion sequence. RT-PCR, Sanger sequencing, and optical genome mapping (OGM) were used to validate the FGs and to determine whether the RARA 5′ and 3′ fusion events were located on the same cistron. The responsiveness of the fusion proteins to ATRA was evaluated using an optimized UAS/GAL4 reporter system. Results Among the cases enrolled, there were 17 males and 6 females, aged 3-66 (median 32) years. Morphological and immunophenotype analyses all showed features of APL. We first identified the STAT3::RARA fusion event in an ATRA-resistant case (the index case, #A1). Moreover, we noticed another fusion event of truncated RARA exon 9 and a 3′ partner sequence that did not map to any annotated protein-coding genes. Further analysis determined it was the RC sequence of the transposable element (TE) LTR40a with the annotated loci at 17p11.2. Both OGM (at the genome level) and RT-PCR (Fig. 1A) confirmed that the two RARA fusion events were in the same cistron. Thus, the fusion transcript being STAT3::RARA::LTR40a-RC spliced in tandem, but not separate transcripts of STAT3::RARA and RARA::LTR40a-RC. We named this novel form of tandem splicingtrinary fusion. We, therefore, recruit X::RARA-APL cases to investigate the recurrence of RARA 3′ fusion. We identified 9 RARA 5′ partner genes in 23 cases (Fig. 1B), with TTMV -ORF2, STAT5B, STAT3, IRF2BP1, THRAP3, and TNRC18 in multiple cases. Ten (43.5%) cases also harbor RARA 3′ fusion partners. The partner of case #A1_8 were TEs, and the partner of case #A9_10 was the same gene as their 5′ one. Thirteen cases did not harbor 3′ fusions. Notably, both TNRC18::RARA cases (#15_16) carry mutations on their RARA ligand binding domain (LBD) at the primary onset. Gene expression analysis confirmed the aberrant activation of the involved TEs. All TEs involved in the RARA 3' fusions confer a poly_A signal sequence, which was essential for a mRNA. In-depth analysis indicated that TEs participate in forming RARA-FGs through a transposition mechanism rather than a translocation mechanism. The molecular events addition to RARA 5′ splicing showed a 5′ partner associated pattern: all of the 4 STAT3::RARA and 4 STAT5B::RARA cases harbor TEs as their 3′ partner, both TNRC18::RARA cases carry LBD mutation, while all 7 TTMV -ORF2::RARA cases did not harbor 3′ splicing or LBD mutation. Remarkably, although the RARA 3' splicing sites varied, they all resulted in partial or complete exon 9 truncation. RARA exon 9 encodes helix 11_12 (H11_12) of its LBD, which is pivotal in allosteric response to ATRA. The RARA trinary fusion will result in LBD-H12 or H11_12 castration due to the 3' splicing, then lead to losing responsiveness to ATRA via an allosteric disability mechanism (Fig. 1C, 1D). Experiments confirmed that cells transfected with STAT3::RARA fusion with intact LBD responded well to ATRA, while both the fusion in case #A1 and artificial fusion with LBD-H12 castration showed no response to physiological and pharmacological concentrations of ATRA (Fig. 1E). Conclusions This is the first report of pathological trinary fusion, the TEs and transposition mechanism participating in composing pathological fusion genes, and the RARA LBD castration confers ATRA resistance in X::RARA-APL. Also, there might be another synergistic mechanism of RARA 5' fusion and LBD mutation. The formation of trinary fusion and gain of LBD mutation, in addition to X::RARA fusion, requires more genome splicing or transposition or mutation events, which explains the rarity of X::RARA-APL. This study highlights mechanisms of partner gene-associated trinary fusion and the orchestrating of multiple molecular events in the leukemogenesis in X::RARA-APL cases.

Trinary Fusion with Ligand Binding Domain Castration Is Crucial in Rarg-Driven APL and Render Unresponsive to ATRA Via Allosteric Disability Mechanism

Introduction All-trans retinoic acid (ATRA) has been used with great success in acute promyelocytic leukemia (APL) cases with PML::RARA fusion gene (FG). There are still PML::RARA negative cases that manifest as APL, and they often carry FGs involved in RARA, RARB, or RARG. X::RARG positive APL ( RARG-APL) cases have been increasingly reported in recent years, thanks to the utilization of transcriptome sequencing (RNA-seq) and whole genome sequencing (WGS). Almost all evaluable RARG-APL cases were resistant to ATRA, and no mechanism has been elucidated. Intriguingly, cells transformed by artificial X::RARG were extremely sensitive to ATRA (PMID: 25510432), indicating intricate mechanisms unrevealed. This study first and systematically unveils the distinctive features and pivotal molecular mechanism of RARG-driven APL. Methods A total of 22 RARG-APL cases with RNA-seq and WGS data from 11 centers were enrolled (Fig. 1A). We used Arriba for routine FG calling and manually investigated the non-coding fusion sequence. Reverse transcription PCR (RT-PCR) and Sanger sequencing were used to validate the FGs and to determine whether the RARG 5′ and 3′ fusion events were located on the same cistron. The responsiveness of the fusion proteins to ATRA was evaluated using an optimized UAS/GAL4 reporter system. Results Among the cases enrolled, there were 15 males and 7 females, aged 0.9-69 (median 38) years. Morphological and immunophenotype analyses all showed features of APL. Routine FGs calling reported RARG fusion to a 5′ partner in each case, with variable RARG splicing sites (Fig. 1A). There were 7 RARG 5' partner genes, with CPSF6, HNRNPC, and NUP98 observed in multiple cases. In the 3 index cases, we identified that they all had RARG 3‘ fusion events besides the 5‘ one (Fig. 1A). RT-PCR confirmed that the 5′ and 3′ RARG fusion events were in the same cistron in each case. Such as, the fusion transcript is the insertion of RARG-e4_9 (e refers to exon) between NPM1-e4 and e11 in case #G1, rather than two separate transcripts of NPM1-e4:: RARG-e4 and RARG-9:: NPM1-e11. We named this novel form of tandem splicingtrinary fusion. Analysis of the 19 validation cases further confirmed that all RARG-APL cases had RARG 3′ fusion events (Fig. 1A). We also validated that the 5′ and 3′ RARG fusion events were in the same cistron in 3 more cases, of which archived cDNA were available. Remarkably, the 3' splicing sits of all cases were consistently at the end of RARG-e9, leading to RARG-e10 truncation. The RARG 3' partner gene was the same as its 5' partner in 11 cases. But in the other 11 cases, the RARG 3' partner was a transposon element (TE) sequence, the most common being a LINE-L2a (8 cases). Sequences analysis indicated that the committed locus of the involved LINE-L2a was at 4.2 Kbp upstream of the RARG gene. Gene expression analysis confirmed the aberrant activation of the involved TEs. All TEs involved in the RARG 3' fusions confer a poly_A signal sequence to the fusion transcript, which was essential for a mRNA. In-depth analysis indicated that TEs participate in the formation of RARG-FGs through a transposition mechanism rather than a translocation mechanism. RARG-e10 encodes helix 11_12 (H11_12) of its ligand binding domain (LBD), which plays a pivotal role in allosteric response to ATRA (PMID: 31178221). The trinary fusion protein with RARG LBD-H11_12 castration will lose responsiveness to ATRA via an allosteric disability mechanism (Fig. 1B, 1C). Experiments confirmed that cells transfected with X::RARG fusion with intact LBD responded well to ATRA, while fusion with LBD-H11_12 castration showed no response to physiological and pharmacological concentrations of ATRA (Fig. 1D, 1E). Conclusions This study clearly revealed that trinary fusion and LBD castration are crucial molecular etiologies in RARG-APL. The trinary fusion orchestrates the aberrant activation of RARG (5‘ splicing) and the complete allosteric disability in responding to ATRA (3‘ splicing). Thus, this study elegantly explains the mechanism of leukemogenesis and the extensive ATRA resistance of RARG-APL. The formation of trinary fusion requires two steps of genome splicing or transposition events, which explains the rarity of RARG-APL. This also clarifies that RARG-APL has a significant disparity in molecular mechanism from PML::RARA-positive APL, and we suggest that it should be considered as a separate entity of acute myeloid leukemia.

Molecular mechanism of regulation of RhoA GTPase by phosphorylation of RhoGDI

Rho-specific guanine nucleotide dissociation inhibitors (RhoGDIs) play a crucial role in the regulation of Rho family GTPases. They act as negative regulators that prevent the activation of Rho GTPases by forming complexes with the inactive GDP-bound state of GTPase. Release of Rho GTPase from the RhoGDI-bound complex is necessary for Rho GTPase activation. Biochemical studies provide evidence of a "phosphorylation code," where phosphorylation of some specific residues of RhoGDI selectively releases its GTPase partner (RhoA, Rac1, Cdc42, etc.). This work attempts to understand the molecular mechanism behind this specific phosphorylation-induced reduction in binding affinity. Using several microseconds long atomistic molecular dynamics simulations of the wild-type and phosphorylated states of the RhoA-RhoGDI complex, we propose a molecular-interaction-based mechanistic model for the dissociation of the complex. Phosphorylation induces major structural changes, particularly in the positively charged polybasic region (PBR) of RhoA and the negatively charged N-terminal region of RhoGDI that contribute most to the binding affinity. Molecular mechanics Poisson-Boltzmann surface area binding energy calculations show a significant weakening of interaction on phosphorylation at the RhoA-specific site of RhoGDI. In contrast, phosphorylation at a Rac1-specific site does not affect the overall binding affinity significantly, which confirms the presence of a phosphorylation code. RhoA-specific phosphorylation leads to a reduction in the number of contacts between the PBR of RhoA and the N-terminal region of RhoGDI, which manifests a reduction of the binding affinity. Using hydrogen bond occupancy analysis and energetic perturbation network, we propose a mechanistic model for the allosteric response, i.e., long-range signal propagation from the site of phosphorylation to the PBR and buried geranylgeranyl group in the form of rearrangement and rewiring of hydrogen bonds and salt bridges. Our results highlight the crucial role of specific electrostatic interactions in manifestation of the phosphorylation code.

Insights into the Allosteric Response to Acidity by the Helicobacter pylori NikR Transcription Factor.

Helicobacter pylori NikR (HpNikR) is a nickel-responsive transcription factor that regulates genes involved in nickel homeostasis, which is essential for the survival of this pathogen within the acidic human stomach. HpNikR also responds to drops in pH and regulates genes controlling acid acclimation of the bacteria, independently of nickel. We previously showed that nickel binding biases the conformational ensemble of HpNikR to the more DNA-binding competent states via an allosteric network of residues encompassing the nickel binding sites and the interface between the metal- and DNA-binding domains. Here, we examine how acidity promotes this response using 19F-NMR, mutagenesis, and DNA-binding studies. 19F-NMR revealed that a drop in pH from 7.6 to 6.0 does little to shift the conformational ensemble of HpNikR to the DNA binding-compatible cis conformer. Nevertheless, DNA-binding affinities of apo-HpNikR at pH 6.0 and Ni(II)-HpNikR at pH 7.6 are comparable for the ureA promoter. Histidine residues of the nickel binding sites were shown to be important for pH-dependent DNA binding and thus likely impart positive charge to the protein, initiating long-range electrostatic interactions with DNA that induce DNA complexation. The results point to a different DNA-binding mechanism in response to acidity compared to the conformational selection mechanism in response to nickel and overall provide new insights into the influence of pH on HpNikR activity, which contributes to H. pylori viability.

Allosteric Determinants in High Temperature Requirement A Enzymes Are Conserved and Regulate the Population of Active Conformations.

High temperature requirement A (HtrA) are allosterically regulated enzymes wherein effector binding to the PDZ domain triggers proteolytic activity. Yet, it remains unclear if the inter-residue network governing allostery is conserved across HtrA enzymes. Here, we investigated and identified the inter-residue interaction networks by molecular dynamics simulations on representative HtrA proteases, Escherichia coli DegS and Mycobacterium tuberculosis PepD, in effector-bound and free forms. This information was used to engineer mutations that could potentially perturb allostery and conformational sampling in a different homologue, M. tuberculosis HtrA. Mutations in HtrA perturbed allosteric regulation─a finding consistent with the hypothesis that the inter-residue interaction network is conserved across HtrA enzymes. Electron density from data collected on cryo-protected HtrA crystals revealed that mutations altered the topology of the active site. Ensemble models fitted into electron density calculated from room-temperature diffraction data showed that only a fraction of these models had a catalytically competent active site conformation alongside a functional oxyanion hole thus providing experimental evidence that these mutations influenced conformational sampling. Mutations at analogous positions in the catalytic domain of DegS perturbed the coupling between effector binding and proteolytic activity, thus confirming the role of these residues in the allosteric response. The finding that a perturbation in the conserved inter-residue network alters conformational sampling and the allosteric response suggests that an ensemble allosteric model best describes regulated proteolysis in HtrA enzymes.

Turning up the heat mimics allosteric signaling in imidazole-glycerol phosphate synthase

Allosteric drugs have the potential to revolutionize biomedicine due to their enhanced selectivity and protection against overdosage. However, we need to better understand allosteric mechanisms in order to fully harness their potential in drug discovery. In this study, molecular dynamics simulations and nuclear magnetic resonance spectroscopy are used to investigate how increases in temperature affect allostery in imidazole glycerol phosphate synthase. Results demonstrate that temperature increase triggers a cascade of local amino acid-to-amino acid dynamics that remarkably resembles the allosteric activation that takes place upon effector binding. The differences in the allosteric response elicited by temperature increase as opposed to effector binding are conditional to the alterations of collective motions induced by either mode of activation. This work provides an atomistic picture of temperature-dependent allostery, which could be harnessed to more precisely control enzyme function.

Introducing NMR strategies to define water molecules that drive metal binding in a transcriptional regulator

Staphylococcus aureus CzrA is a paradigmatic member of the ArsR family of transcriptional metalloregulators, which are critical for the bacterial response to stress. Zinc binding to CzrA, which induces DNA derepression, is entropically driven, as shown by calorimetry. A detailed equilibrium dynamics study of different allosteric states of CzrA revealed that zinc induces an entropy redistribution that controls for DNA binding regulation; however, this change in conformational entropy only accounts for a small net contribution to the total entropy. This difference between the change in conformational entropy vs. total entropy of zinc binding implies a significant contribution of solvent molecule rearrangements to this equilibrium. However, the absence of major structural changes suggests that solvent rearrangements occur mainly on the protein surface and/or from zinc desolvation, concomitant with a dynamical redistribution of conformational entropy. Previous results also suggest that zinc binding not only leads to a redistribution of protein internal dynamics, but also release of water molecules from the protein surface. In turn, these water molecules may make a significant contribution to the allosteric response that results in dissociation from the DNA.Quantifying the differential hydration of two conformational states that share very similar crystal structures and then correlating this with the protein's solvent entropy change constitutes an unresolved problem, even when thermodynamics suggest a significant contribution of solvent entropy. Here, we present different avenues to dissect hydration dynamics in a metal-binding transcriptional regulator that provide different insights into this complex problem. We explore primary solution NMR tools for probing protein–water interactions: the laboratory frame nuclear Overhauser effect (NOE) and its rotating frame counterpart (ROE) between long-lived water molecules and the protein residues. The wNOE/wROE ratio is a promising tool for the detection of hydration dynamics near the surface of a protein in a site-specific manner, minimizing contamination from bulk solvent. Molecular dynamics simulations and computational methods designed to provide a spatially resolved picture of solvent thermodynamics were also employed to provide a more complete panorama of solvent redistribution.

SARS-CoV-2 spike variants differ in their allosteric responses to linoleic acid.

The SARS-CoV-2 spike protein contains a functionally important fatty acid (FA) binding site, which is also found in some other coronaviruses, e.g. SARS-CoV and MERS-CoV. The occupancy of the FA site by linoleic acid (LA) reduces infectivity by 'locking' the spike in a less infectious conformation. Here, we use dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations to compare the allosteric responses of spike variants to LA removal. D-NEMD simulations show that the FA site is coupled to other functional regions of the protein, e.g. the receptor-binding motif (RBM), N-terminal domain (NTD), furin cleavage site, and regions surrounding the fusion peptide. D-NEMD simulations also identify the allosteric networks connecting the FA site to these functional regions. The comparison between the wild-type spike and four variants (Alpha, Delta, Delta plus, and Omicron BA.1) shows that the variants differ significantly in their responses to LA removal. The allosteric connections to the FA site on Alpha are generally similar to those on the wild-type protein, with the exception of the RBM and the S71-R78 region, which show a weaker link to the FA site. In contrast, Omicron is the most different variant, exhibiting significant differences in the RBM, NTD, V622-L629, and furin cleavage site. These differences in the allosteric modulation may be of functional relevance, potentially affecting transmissibility and virulence. Experimental comparison of the effects of LA on SARS-CoV-2 variants, including emerging variants, is warranted.

New TSPO Crystal Structures of Mutant and Heme-Bound Forms with Altered Flexibility, Ligand Binding, and Porphyrin Degradation Activity.

The ancient protein TSPO (translocator protein 18kD) is found in all kingdoms and was originally identified as a binding site of benzodiazepine drugs. Its physiological function remains unclear, although porphyrins are conserved ligands. Several crystal structures of bacterial TSPO and nuclear magnetic resonance structures of a mouse form have revealed monomer and dimer configurations, but there have been no reports of structures with a physiological ligand. Here, we present the first X-ray structures of Rhodobacter sphaeroides TSPO with a physiological ligand bound. Two different variants (substituting threonine for alanine at position 139 (A139T) and phenylalanine for alanine at position 138 (A138F)) yielded well-diffracting crystals giving structures of both apo- and heme-containing forms. Both variants have wild-type micromolar affinity for heme and protoporphyrin IX, but A139T has very low ability to accelerate the breakdown of porphyrin in the presence of light and oxygen. The binding of heme to one protomer of the dimer of either mutant induces a more rigid structure, both in the heme-binding protomer and the protomer without heme bound, demonstrating an allosteric response. Ensemble refinement of the X-ray data reveals distinct regions of altered flexibility in response to single heme binding to the dimer. The A139T variant shows a more rigid structure overall, which may relate to extra hydrogen bonding of waters captured in the heme crevice. As TSPO has been suggested to have a role in heme delivery from mitochondria to the cytoplasm, the new structures provide potential clues regarding the structural basis of such activity.

Room-temperature crystallography reveals altered binding of small-molecule fragments to PTP1B.

Much of our current understanding of how small-molecule ligands interact with proteins stems from X-ray crystal structures determined at cryogenic (cryo) temperature. For proteins alone, room-temperature (RT) crystallography can reveal previously hidden, biologically relevant alternate conformations. However, less is understood about how RT crystallography may impact the conformational landscapes of protein-ligand complexes. Previously, we showed that small-molecule fragments cluster in putative allosteric sites using a cryo crystallographic screen of the therapeutic target PTP1B (Keedy et al., 2018). Here, we have performed two RT crystallographic screens of PTP1B using many of the same fragments, representing the largest RT crystallographic screens of a diverse library of ligands to date, and enabling a direct interrogation of the effect of data collection temperature on protein-ligand interactions. We show that at RT, fewer ligands bind, and often more weakly - but with a variety of temperature-dependent differences, including unique binding poses, changes in solvation, new binding sites, and distinct protein allosteric conformational responses. Overall, this work suggests that the vast body of existing cryo-temperature protein-ligand structures may provide an incomplete picture, and highlights the potential of RT crystallography to help complete this picture by revealing distinct conformational modes of protein-ligand systems. Our results may inspire future use of RT crystallography to interrogate the roles of protein-ligand conformational ensembles in biological function.

From elucidating mechanisms of allosteric signaling to designing allosteric drugs and biologics.

Allostery is a pervasive phenomenon underlies a multitude of cellular processes. We have previously developed a Structure-Based Statistical Mechanical Model of Allostery (SBSMMA) to quantitatively describe the causality and energetics of allosteric signaling due to perturbations such as ligand binding, mutations, disorder-order transition and post-translational modifications. The computational model was implemented in the AlloSigMA server and AlloMAPS database, which allow one to explore allosteric effects of perturbations in form of the exhaustive Allosteric Signaling/Probing Map (ASM/APM), to identify potential allosteric sites, and to facilitate effector design. Here, we demonstrate some recent work on applying our broadly applicable framework to different aspects of allostery. First, analysis of communication between different sites/regions of the Spike protein from SARS-CoV-2 revealed mutations harbored by variants of concern, as well as glycosylation, that originate strong signaling to the distant receptor-binding domain despite the separation. Using reverse perturbation of allosteric communication and ASM/APM, we identified potential druggable sites that can induce tunable allosteric responses in different functional regions of Spike. Second, while the strengths of allosteric medicines are widely recognized, rational design of effectors remains a challenging problem. We demonstrated with GPCRs and kinases that effector design should begin with the search/design for latent/non-natural binding sites, followed by fragment-based design of ligands, and consequently mutual adjustments of site-ligand pairs to obtain and tune the desired binding affinity and allosteric signals. Third, rapid progress in harnessing allostery calls for an understanding of how communication is established by different structural elements and how it can be modified. We obtained a picture of conserved allosteric communication characteristic in major folds, alterations of structure-driven signaling via sequence-determined divergence to distinct functions, and its diversification in multi-domain proteins and oligomers, which will facilitate engineering/design of allosteric communication.

Reprogrammable allosteric metamaterials from disordered networks.

Prior works on disordered mechanical metamaterial networks-consisting of fixed nodes connected by discrete bonds-have shown that auxetic and allosteric responses can be achieved by pruning a specific set of the bonds from an originally random network. However, bond pruning is irreversible and yields a single bulk response. Using material stiffness as a tunable design parameter, we create metamaterial networks where allosteric responses are achieved without bond removal. Such systems are experimentally realized through variable stiffness bonds that can strengthen and weaken on-demand. In a disordered mechanical network with variable stiffness bonds, different subsets of bonds can be strategically softened to achieve different bulk responses, enabling a multiplicity of reprogrammable input/output allosteric responses.

Mechanistic insights into the nickel-dependent allosteric response of the Helicobacter pylori NikR transcription factor

In Helicobacter pylori, the nickel-responsive NikR transcription factor plays a key role in regulating intracellular nickel concentrations, which is an essential process for survival of this pathogen in the acidic human stomach. Nickel binding to H.pylori NikR (HpNikR) allosterically activates DNA binding to target promoters encoding genes involved in nickel homeostasis and acid adaptation, to either activate or repress their transcription. We previously showed that HpNikR adopts an equilibrium between an open conformation and DNA-binding competent cis and trans states. Nickel binding slows down conformational exchange between these states and shifts the equilibrium toward the binding-competent states. The protein then becomes stabilized in a cis conformation upon binding the ureA promoter. Here, we investigate how nickel binding creates this response and how it is transmitted to the DNA-binding domains. Through mutagenesis, DNA-binding studies, and computational methods, the allosteric response to nickel was found to be propagated from the nickel-binding sites to the DNA-binding domains via the β-sheets of the metal-binding domain and a network of residues at the inter-domain interface. Our computational results suggest that nickel binding increases protein rigidity to slow down the conformational exchange. A thymine base in the ureA promoter sequence, known to be critical for high affinity DNA binding by HpNikR, was also found to be important for the allosteric response, while a modified version of this promoter further highlighted the importance of the DNA sequence in modulating the response. Collectively, our results provide insights into regulation of a key protein for H.pylori survival.

Conformational interdomain flexibility in a bacterial α-isopropylmalate synthase is necessary for leucine biosynthesis

α-Isopropylmalate synthase (IPMS) catalyzes the first step in leucine (Leu) biosynthesis and is allosterically regulated by the pathway end product, Leu. IPMS is a dimeric enzyme with each chain consisting of catalytic, accessory, and regulatory domains, with the accessory and regulatory domains of each chain sitting adjacent to the catalytic domain of the other chain. The IPMS crystal structure shows significant asymmetry due to different relative domain conformations in each chain. Owing to the challenges posed by the dynamic and asymmetric structures of IPMS enzymes, the molecular details of their catalytic and allosteric mechanisms are not fully understood. In this study, we have investigated the allosteric feedback mechanism of the IPMS enzyme from the bacterium that causes meningitis, Neisseria meningitidis (NmeIPMS). By combining molecular dynamics (MD) simulations with small-angle X-ray scattering (SAXS), mutagenesis, and heterodimer-generation, we demonstrate that Leu-bound NmeIPMS is in a rigid conformational state stabilized by asymmetric inter-domain polar interactions. Furthermore, we found removing these polar interactions by mutagenesis impaired the allosteric response without compromising Leu binding. Our results suggest that the allosteric inhibition of NmeIPMS is achieved by restricting the flexibility of the accessory and regulatory domains, demonstrating that significant conformational flexibility is required for catalysis.

In Vitro Characterization of a Nuclear Receptor-like Domain of the Xylanase Regulator 1 from Trichoderma reesei.

Engineering transcription factors is an interesting research target gaining increasing attention, such as in the case of industrially used organisms. With respect to sustainability, biomass-degrading saprophytic fungi, such as Trichoderma reesei, are promising industrial work horses because they exhibit a high secretory capacity of native and heterologously expressed enzymes and compounds. A single-point mutation in the main transactivator of xylanase and cellulase expressions in T. reesei Xyr1 led to a strongly deregulated and enhanced xylanase expression. Circular dichroism spectroscopy revealed a change in secondary structure caused by this mutation. According to electrophoretic mobility shift assays and determination of the equilibrium-binding constants, the DNA-binding affinity of the mutated Xyr1 was considerably reduced compared to the wild-type Xyr1. Both techniques were also used to investigate the allosteric response to carbohydrates (D-glucose-6-phosphate, D-xylose, and sophorose) signalling the repression or induction of Xyr1 target genes. The mutated Xyr1 no longer exhibited a conformational change in response to these carbohydrates, indicating that the observed deregulation is not a simple matter of a change in DNA-binding of the transactivator. Altogether, we postulate that the part of Xyr1 where the mutation is located functions as a nuclear receptor-like domain that mediates carbohydrate signals and modulates the Xyr1 transactivating activity.