Induced-fit Mechanism Research Articles

Exploration of Cryptic Pockets Using Enhanced Sampling Along Normal Modes: A Case Study of KRAS G12D.

Identification of cryptic pockets has the potential to open new therapeutic opportunities by discovering ligand binding sites that remain hidden in static apo structures of a target protein. Moreover, allosteric cryptic pockets can become valuable for designing target-selective ligands when the natural ligand binding sites are conserved in variants of a protein. For example, before an allosteric cryptic pocket was discovered, KRAS was considered undruggable due to its smooth surface and conservation of the GDP/GTP binding pocket across the wild type and oncogenic isoforms. Recent identification of the Switch-II cryptic pocket in the KRASG12C mutant and FDA approval of anticancer drugs targeting this site underscores the importance of cryptic pockets in solving pharmaceutical challenges. Here, we present a newly developed approach for the exploration of cryptic pockets using weighted ensemble molecular dynamics simulations with inherent normal modes as progress coordinates applied to the wild type KRAS and the G12D mutant. We performed extensive all-atomic simulations (>400 μs) with and without several cosolvents (xenon, ethanol, benzene), and analyzed trajectories using three distinct methods to search for potential binding pockets. These methods have been applied as a proof-of-concept to KRAS and have shown they can predict known cryptic binding sites. Furthermore, we performed ligand-binding simulations of a known inhibitor (MRTX1133) to shed light on the nature of cryptic pockets in KRASG12D and the role of conformational selection vs induced-fit mechanism in the formation of these cryptic pockets.

On the function of TRAP substrate-binding proteins: Conformational variation of the sialic acid binding protein SiaP

Tripartite ATP-independent periplasmic (TRAP) transporters are analogous to ABC transporters in that they use a substrate-binding protein to scavenge metabolites (e.g., N-acetylneuraminate) and deliver them to the membrane components for import. TRAP substrate-binding proteins are thought to bind the substrate using a two-state (open and closed) induced-fit mechanism. We solved the structure of the TRAP N-acetylneuraminate substrate-binding protein from Aggregatibacter actinomycetemcomitans (AaSiaP) in both the open ligand-free and closed liganded conformations. Surprisingly, we also observed an intermediate conformation, where AaSiaP is mostly closed and is bound to a non-cognate ligand, acetate, which hints at how N-acetylneuraminate binding stabilises a fully closed state. AaSiaP preferentially binds N-acetylneuraminate (KD = 0.4 μM) compared to N-glycolylneuraminate (KD = 4.4 μM), which is explained by the closed-N-acetylneuraminate bound structure. Small-angle X-ray scattering data alongside molecular dynamics simulations suggest the AaSiaP adopts a more open state in solution than in crystal. However, the open unliganded conformation can also sample closed conformations. Molecular dynamics simulations also demonstrate the importance of water molecules for stabilising the closed conformation. Although our data is consistent with an induced fit model of binding, we suggest that the open unliganded conformation may sample multiple states capable of binding substrate. The mechanism by which the ligand is released for import remains to be determined.

Conformational Modulation of a Mobile Loop Controls Catalysis in the (βα)8-Barrel Enzyme of Histidine Biosynthesis HisF.

The overall significance of loop motions for enzymatic activity is generally accepted. However, it has largely remained unclear whether and how such motions can control different steps of catalysis. We have studied this problem on the example of the mobile active site β1α1-loop (loop1) of the (βα)8-barrel enzyme HisF, which is the cyclase subunit of imidazole glycerol phosphate synthase. Loop1 variants containing single mutations of conserved amino acids showed drastically reduced rates for the turnover of the substrates N'-[(5'-phosphoribulosyl) formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PrFAR) and ammonia to the products imidazole glycerol phosphate (ImGP) and 5-aminoimidazole-4-carboxamide-ribotide (AICAR). A comprehensive mechanistic analysis including stopped-flow kinetics, X-ray crystallography, NMR spectroscopy, and molecular dynamics simulations detected three conformations of loop1 (open, detached, closed) whose populations differed between wild-type HisF and functionally affected loop1 variants. Transient stopped-flow kinetic experiments demonstrated that wt-HisF binds PrFAR by an induced-fit mechanism whereas catalytically impaired loop1 variants bind PrFAR by a simple two-state mechanism. Our findings suggest that PrFAR-induced formation of the closed conformation of loop1 brings active site residues in a productive orientation for chemical turnover, which we show to be the rate-limiting step of HisF catalysis. After the cyclase reaction, the closed loop conformation is destabilized, which favors the formation of detached and open conformations and hence facilitates the release of the products ImGP and AICAR. Our data demonstrate how different conformations of active site loops contribute to different catalytic steps, a finding that is presumably of broad relevance for the reaction mechanisms of (βα)8-barrel enzymes and beyond.

Thermodynamic compensation to temperature extremes in B. subtilis vs T. maritima lysine riboswitches

T. maritima and B. subtilis are bacteria that inhabit significantly different thermal environments, ∼80 vs. ∼40°C, yet employ similar lysine riboswitches to aid in the transcriptional regulation of the genes involved in the synthesis and transport of amino acids. Despite notable differences in G-C basepair frequency and primary sequence, the aptamer moieties of each riboswitch have striking similarities in tertiary structure, with several conserved motifs and long-range interactions. To explore genetic adaptation in extreme thermal environments, we compare the kinetic and thermodynamic behaviors in T. maritima and B. subtilis lysine riboswitches via single-molecule fluorescence resonance energy transfer analysis. Kinetic studies reveal that riboswitch folding rates increase with lysine concentration while the unfolding rates are independent of lysine. This indicates that both riboswitches bind lysine through an induced-fit (“bind-then-fold”) mechanism, with lysine binding necessarily preceding conformational changes. Temperature-dependent van’t Hoff studies reveal qualitative similarities in the thermodynamic landscapes for both riboswitches in which progression from the open, lysine-unbound state to both transition states (‡) and closed, lysine-bound conformations is enthalpically favored yet entropically penalized, with comparisons of enthalpic and entropic contributions extrapolated to a common [K+] = 100 mM in quantitative agreement. Finally, temperature-dependent Eyring analysis reveals the TMA and BSU riboswitches to have remarkably similar folding/unfolding rate constants when extrapolated to their respective (40 and 80°C) environmental temperatures. Such behavior suggests a shared strategy for ligand binding and aptamer conformational change in the two riboswitches, based on thermodynamic adaptations in number of G-C basepairs and/or modifications in tertiary structure that stabilize the ligand-unbound conformation to achieve biocompetence under both hyperthermophilic and mesothermophilic conditions.

Docking, MD Simulations, and DFT Calculations: Assessing W254's Function and Sartan Binding in Furin.

Furins are serine endoproteases that are involved in many biological processes, where they play important roles in normal metabolism, in the activation of various pathogens, while they are a target for therapeutic intervention. Dichlorophenyl-pyridine "BOS" compounds are well known drugs that are used as inhibitors of human furin by an induced-fit mechanism, in which tryptophan W254 in the furin catalytic cleft acts as a molecular transition energy gate. The binding of "BOS" drug into the active center of furin has been computationally studied using the density functional theory (DFT) and ONIOM multiscaling methodologies. The binding enthalpies of the W254 with the furin-BOS is -32.8 kcal/mol ("open") and -18.8 kcal/mol ("closed"), while the calculated torsion barrier was found at 30 kcal/mol. It is significantly smaller than the value of previous MD calculations due to the relaxation of the environment, i.e., nearby groups of the W254, leading to the reduction of the energy demands. The significant lower barrier explains the experimental finding that the dihedral barrier of W254 is overcome. Furthermore, sartans were studied to evaluate their potential as furin inhibitors. Sartans are AT1 antagonists, and they effectively inhibit the hypertensive effects induced by the peptide hormone Angiotensin II. Here, they have been docked into the cavity to evaluate their effect on the BOS ligand via docking and molecular dynamics simulations. A consistent binding of sartans within the cavity during the simulation was found, suggesting that they could act as furin inhibitors. Finally, sartans interact with the same amino acids as W254, leading to a competitive binding that may influence the pharmacological efficacy and potential drug interactions of sartans.

Impact of Nonnative Interactions on the Binding Kinetics of Intrinsically Disordered p53 with MDM2: Insights from All-Atom Simulation and Markov State Model Analysis.

Intrinsically disordered proteins (IDPs) lack a well-defined tertiary structure but are essential players in various biological processes. Their ability to undergo a disorder-to-order transition upon binding to their partners, known as the folding-upon-binding process, is crucial for their function. One classical example is the intrinsically disordered transactivation domain (TAD) of the tumor suppressor protein p53, which quickly forms a structured α-helix after binding to its partner MDM2, with clinical significance for cancer treatment. However, the contribution of nonnative interactions between the IDP and its partner to the rapid binding kinetics, as well as their interplay with native interactions, is not well understood at the atomic level. Here, we used molecular dynamics simulation and Markov state model (MSM) analysis to study the folding-upon-binding mechanism between p53-TAD and MDM2. Our results suggest that the system progresses from the nascent encounter complex to the well-structured encounter complex and finally reaches the native complex, following an induced-fit mechanism. We found that nonnative hydrophobic and hydrogen bond interactions, combined with native interactions, effectively stabilize the nascent and well-structured encounter complexes. Among the nonnative interactions, Leu25p53-Leu54MDM2 and Leu25p53-Phe55MDM2 are particularly noteworthy, as their interaction strength is close to the optimum. Evidently, strengthening or weakening these interactions could both adversely affect the binding kinetics. Overall, our findings suggest that nonnative interactions are evolutionarily optimized to accelerate the binding kinetics of IDPs in conjunction with native interactions.

The Streptococcus phage protein paratox is an intrinsically disordered protein.

The bacteriophage protein paratox (Prx) blocks quorum sensing in its streptococcal host by directly binding the signal receptor and transcription factor ComR. This reduces the ability of Streptococcus to uptake environmental DNA and protects phage DNA from damage by recombination. Past work characterizing the Prx:ComR molecular interaction revealed that paratox adopts a well-ordered globular fold when bound to ComR. However, solution-state biophysical measurements suggested that Prx may be conformationally dynamic. To address this discrepancy, we investigated the stability and dynamic properties of Prx in solution using circular dichroism, nuclear magnetic resonance, and several fluorescence-based protein folding assays. Our work shows that under dilute buffer conditions Prx is intrinsically disordered. We also show that the addition of kosmotropic salts or protein stabilizing osmolytes induces Prx folding. However, the solute stabilized fold is different from the conformation Prx adopts when it is bound to ComR. Furthermore, we have characterized Prx folding thermodynamics and folding kinetics through steady-state fluorescence and stopped flow kinetic measurements. Our results show that Prx is a highly dynamic protein in dilute solution, folding and refolding within the 10 ms timescale. Overall, our results demonstrate that the streptococcal phage protein Prx is an intrinsically disordered protein in a two-state equilibrium with a solute-stabilized folded form. Furthermore, the solute-stabilized fold is likely the predominant form of Prx in a solute-crowded bacterial cell. Finally, our work suggests that Prx binds and inhibits ComR, and thus quorum sensing in Streptococcus, by a combination of conformational selection and induced-fit binding mechanisms.

Dishevelled2 activates WGEF via its interaction with a unique internal peptide motif of the GEF

The Wnt-planar cell polarity (Wnt-PCP) pathway is crucial in establishing cell polarity during development and tissue homoeostasis. This pathway is found to be dysregulated in many pathological conditions, including cancer and autoimmune disorders. The central event in Wnt-PCP pathway is the activation of Weak-similarity guanine nucleotide exchange factor (WGEF) by the adapter protein Dishevelled (Dvl). The PDZ domain of Dishevelled2 (Dvl2PDZ) binds and activates WGEF by releasing it from its autoinhibitory state. However, the actual Dvl2PDZ binding site of WGEF and the consequent activation mechanism of the GEF have remained elusive. Using biochemical and molecular dynamics studies, we show that a unique “internal-PDZ binding motif” (IPM) of WGEF mediates the WGEF-Dvl2PDZ interaction to activate the GEF. The residues at P2, P0, P-2 and P-3 positions of IPM play an important role in stabilizing the WGEFpep-Dvl2PDZ interaction. Furthermore, MD simulations of modelled Dvl2PDZ-WGEFIPM peptide complexes suggest that WGEF-Dvl2PDZ interaction may differ from the reported Dvl2PDZ-IPM interactions. Additionally, the apo structure of human Dvl2PDZ shows conformational dynamics different from its IPM peptide bound state, suggesting an induced fit mechanism for the Dvl2PDZ-peptide interaction. The current study provides a model for Dvl2 induced activation of WGEF.

Discovery of a small-molecule inhibitor that traps Polθ on DNA and synergizes with PARP inhibitors

The DNA damage response (DDR) protein DNA Polymerase θ (Polθ) is synthetic lethal with homologous recombination (HR) factors and is therefore a promising drug target in BRCA1/2 mutant cancers. We discover an allosteric Polθ inhibitor (Polθi) class with 4–6 nM IC50 that selectively kills HR-deficient cells and acts synergistically with PARP inhibitors (PARPi) in multiple genetic backgrounds. X-ray crystallography and biochemistry reveal that Polθi selectively inhibits Polθ polymerase (Polθ-pol) in the closed conformation on B-form DNA/DNA via an induced fit mechanism. In contrast, Polθi fails to inhibit Polθ-pol catalytic activity on A-form DNA/RNA in which the enzyme binds in the open configuration. Remarkably, Polθi binding to the Polθ-pol:DNA/DNA closed complex traps the polymerase on DNA for more than forty minutes which elucidates the inhibitory mechanism of action. These data reveal a unique small-molecule DNA polymerase:DNA trapping mechanism that induces synthetic lethality in HR-deficient cells and potentiates the activity of PARPi.

Toward More Selective Antibiotic Inhibitors: A Structural View of the Complexed Binding Pocket of E. coli Peptide Deformylase.

Peptide deformylase (PDF) is involved in bacterial protein maturation processes. Originating from the interest in a new antibiotic, tremendous effort was put into the refinement of PDF inhibitors (PDFIs) and their selectivity. We obtained a full NMR backbone assignment the emergent additional protein backbone resonances of ecPDF 1-147 in complex with 2-(5-bromo-1H-indol-3-yl)-N-hydroxyacetamide (2), a potential new structural scaffold for more selective PDFIs. We also determined the complex crystal structures of E. coli PDF (ecPDF fl) and 2. Our structure suggests an alternative ligand conformation within the protein, a possible starting point for further selectivity optimization. The orientation of the second ligand conformation in the crystal structure points toward a small region of the S1' pocket, which differs between bacterial PDFs and human PDF. Moreover, we analyzed the binding mode of 2 via NMR TITAN line shape analysis, revealing an induced fit mechanism.

Kinetics of Intermediate Release Enhances P450 11B2-Catalyzed Aldosterone Synthesis.

The mitochondrial enzyme cytochrome P450 11B2 (aldosterone synthase) catalyzes the 3 terminal transformations in the biosynthesis of aldosterone from 11-deoxycorticosterone (DOC): 11β-hydroxylation to corticosterone, 18-hydroxylation, and 18-oxidation. Prior studies have shown that P450 11B2 produces more aldosterone from DOC than from the intermediate corticosterone and that the reaction sequence is processive, with intermediates remaining bound to the active site between oxygenation reactions. In contrast, P450 11B1 (11β-hydroxylase), which catalyzes the terminal step in cortisol biosynthesis, shares a 93% amino acid sequence identity with P450 11B2, converts DOC to corticosterone, but cannot synthesize aldosterone from DOC. The biochemical and biophysical properties of P450 11B2, which enable its unique 18-oxygenation activity and processivity, yet are not also represented in P450 11B1, remain unknown. To understand the mechanism of aldosterone biosynthesis, we introduced point mutations at residue 320, which partially exchange the activities of P450 11B1 and P450 11B2 (V320A and A320V, respectively). We then investigated NADPH coupling efficiencies, binding kinetics and affinities, and product formation of purified P450 11B1 and P450 11B2, wild-type, and residue 320 mutations in phospholipid vesicles and nanodiscs. Coupling efficiencies for the 18-hydroxylase reaction with corticosterone as the substrate failed to correlate with aldosterone synthesis, ruling out uncoupling as a relevant mechanism. Conversely, corticosterone dissociation rates correlated inversely with aldosterone production. We conclude that intermediate dissociation kinetics, not coupling efficiency, enable P450 11B2 to synthesize aldosterone via a processive mechanism. Our kinetic data also suggest that the binding of DOC to P450 11B enzymes occurs in at least two distinct steps, favoring an induced-fit mechanism.

Fundamental Mechanisms in Membrane Receptology: Old Paradigms, New Concepts and Perspectives

Receptology, the science of receptors, is a multidimensional field of research which can be dissected into biosynthesis, membrane sorting, ligand binding and signal transduction. Plasma membrane receptors connect the cells with their environment and transmit signals that are translated into biological information. The historical paradigm of ligand–receptor interactions is the lock-and-key model. This model presupposes that both partners have a precise 3D shape that perfectly fits together to form the ligand–receptor complex. However, this simple model suffers from severe limitations due to several levels of simplifications: (i) water molecules and membrane lipids are not considered; (ii) not all ligands have a stable 3D structure; (iii) the ligand-binding pocket of the receptor is often flexible and conformationally rearranged after the initial binding step (induced fit mechanism) and/or subjected to conformational selection by the ligand; (iv) there are signal transduction mechanisms which can be either purely mechanical (conformational change of the receptor induced after binding of the ligand), lipid-assisted (e.g., by raft lipids such as cholesterol or gangliosides), or in some instances of quantic nature (detection of odorant molecules). The aim of the present review is to challenge the old paradigms and present new concepts of membrane receptology that consider the impact of critical parameters such as water molecules, membrane lipids, electrostatic surface potential and quantum mechanisms.

Structural Differences at Quadruplex-Duplex Interfaces Enable Ligand-Induced Topological Transitions.

Quadruplex-duplex (QD) junctions, which represent unique structural motifs of both biological and technological significance, have been shown to constitute high-affinity binding sites for various ligands. A QD hybrid construct based on a human telomeric sequence, which harbors a duplex stem-loop in place of a short lateral loop, is structurally characterized by NMR. It folds into two major species with a (3+1) hybrid and a chair-type (2+2) antiparallel quadruplex domain coexisting in a K+ buffer solution. The antiparallel species is stabilized by an unusual capping structure involving a thymine and protonated adenine base AH+ of the lateral loop facing the hairpin duplex to form a T·AH+·G·C quartet with the interfacial G·C base pair at neutral pH. Addition and binding of Phen-DC3 to the QD hybrid mixture by its partial intercalation at corresponding QD junctions leads to a topological transition with exclusive formation of the (3+1) hybrid fold. In agreement with the available experimental data, such an unprecedented discrimination of QD junctions by a ligand can be rationalized following an induced fit mechanism.

Folding-upon-binding pathways of an intrinsically disordered protein from a deep Markov state model

A central challenge in the study of intrinsically disordered proteins is the characterization of the mechanisms by which they bind their physiological interaction partners. Here, we utilize a deep learning-based Markov state modeling approach to characterize the folding-upon-binding pathways observed in a long timescale molecular dynamics simulation of a disordered region of the measles virus nucleoprotein NTAIL reversibly binding the X domain of the measles virus phosphoprotein complex. We find that folding-upon-binding predominantly occurs via two distinct encounter complexes that are differentiated by the binding orientation, helical content, and conformational heterogeneity of NTAIL. We observe that folding-upon-binding predominantly proceeds through a multi-step induced fit mechanism with several intermediates and do not find evidence for the existence of canonical conformational selection pathways. We observe four kinetically separated native-like bound states that interconvert on timescales of eighty to five hundred nanoseconds. These bound states share a core set of native intermolecular contacts and stable NTAIL helices and are differentiated by a sequential formation of native and non-native contacts and additional helical turns. Our analyses provide an atomic resolution structural description of intermediate states in a folding-upon-binding pathway and elucidate the nature of the kinetic barriers between metastable states in a dynamic and heterogenous, or "fuzzy", protein complex.

Structural basis of hepatitis B virus receptor binding.

Hepatitis B virus (HBV), a leading cause of developing hepatocellular carcinoma affecting more than 290 million people worldwide, is an enveloped DNA virus specifically infecting hepatocytes. Myristoylated preS1 domain of the HBV large surface protein binds to the host receptor sodium-taurocholate cotransporting polypeptide (NTCP), a hepatocellular bile acid transporter, to initiate viral entry. Here, we report the cryogenic-electron microscopy structure of the myristoylated preS1 (residues 2-48) peptide bound to human NTCP. The unexpectedly folded N-terminal half of the peptide embeds deeply into the outward-facing tunnel of NTCP, whereas the C-terminal half formed extensive contacts on the extracellular surface. Our findings reveal an unprecedented induced-fit mechanism for establishing high-affinity virus-host attachment and provide a blueprint for the rational design of anti-HBV drugs targeting virus entry.

Excited States of apo-Guanidine-III Riboswitch Contribute to Guanidinium Binding through Both Conformational and Induced-Fit Mechanisms.

Riboswitches are mRNA segments that regulate gene expression through conformational changes driven by their cognate ligand binding. The ykkC motif forms a riboswitch class that selectively senses a guanidinium ion (Gdm+) and regulates the downstream expression of proteins which aid in the efflux of excess Gdm+ from the cells. The aptamer domain (AD) of the guanidine-III riboswitch forms an H-type pseudoknot with a triple helical domain that binds a Gdm+. We studied the binding of Gdm+ to the AD of the guanidine (ykkC)-III riboswitch using computer simulations to probe the specificity of the riboswitch to Gdm+ binding. We show that Gdm+ binding is a fast process occurring on the nanosecond time scale, with minimal conformational changes to the AD. Using machine learning and Markov-state models, we identified the excited conformational states of the AD, which have a high Gdm+ binding propensity, making the Gdm+ binding landscape complex exhibiting both conformational selection and induced-fit mechanisms. The proposed apo-AD excited states and their role in the ligand-sensing mechanism are amenable to experimental verification. Further, targeting these excited-state conformations in discovering new antibiotics can be explored.

Elucidating Dynamics of Adenylate Kinase from Enzyme Opening to Ligand Release.

This study explores ligand-driven conformational changes in adenylate kinase (AK), which is known for its open-to-close conformational transitions upon ligand binding and release. By utilizing string free energy simulations, we determine the free energy profiles for both enzyme opening and ligand release and compare them with profiles from the apoenzyme. Results reveal a three-step ligand release process, which initiates with the opening of the adenosine triphosphate-binding subdomain (ATP lid), followed by ligand release and concomitant opening of the adenosine monophosphate-binding subdomain (AMP lid). The ligands then transition to nonspecific positions before complete dissociation. In these processes, the first step is energetically driven by ATP lid opening, whereas the second step is driven by ATP release. In contrast, the AMP lid opening and its ligand release make minor contributions to the total free energy for enzyme opening. Regarding the ligand binding mechanism, our results suggest that AMP lid closure occurs via an induced-fit mechanism triggered by AMP binding, whereas ATP lid closure follows conformational selection. This difference in the closure mechanisms provides an explanation with implications for the debate on ligand-driven conformational changes of AK. Additionally, we determine an X-ray structure of an AK variant that exhibits significant rearrangements in the stacking of catalytic arginines, explaining its reduced catalytic activity. In the context of apoenzyme opening, the sequence of events is different. Here, the AMP lid opens first while the ATP lid remains closed, and the free energy associated with ATP lid opening varies with orientation, aligning with the reported AK opening and closing rate heterogeneity. Finally, this study, in conjunction with our previous research, provides a comprehensive view of the intricate interplay between various structural elements, ligands, and catalytic residues that collectively contribute to the robust catalytic power of the enzyme.

Unravelling the mechanism of neurotensin recognition by neurotensin receptor 1

The conformational ensembles of G protein-coupled receptors (GPCRs) include inactive and active states. Spectroscopy techniques, including NMR, show that agonists, antagonists and other ligands shift the ensemble toward specific states depending on the pharmacological efficacy of the ligand. How receptors recognize ligands and the kinetic mechanism underlying this population shift is poorly understood. Here, we investigate the kinetic mechanism of neurotensin recognition by neurotensin receptor 1 (NTS1) using 19F-NMR, hydrogen-deuterium exchange mass spectrometry and stopped-flow fluorescence spectroscopy. Our results indicate slow-exchanging conformational heterogeneity on the extracellular surface of ligand-bound NTS1. Numerical analysis of the kinetic data of neurotensin binding to NTS1 shows that ligand recognition follows an induced-fit mechanism, in which conformational changes occur after neurotensin binding. This approach is applicable to other GPCRs to provide insight into the kinetic regulation of ligand recognition by GPCRs.

Substrate-independent activation pathways of the CRISPR-Cas9 HNH nuclease

A hallmark of tightly regulated high-fidelity enzymes is that they become activated only after encountering cognate substrates, often by an induced-fit mechanism rather than conformational selection. Upon analysis of molecular dynamics trajectories, we recently discovered that the Cas9 HNH domain exists in three conformations: 1) Y836 (which is two residues away from the catalytic D839 and H840 residues) is hydrogen bonded to the D829 backbone amide, 2) Y836 is hydrogen bonded to the backbone amide of D861 (which is one residue away from the third catalytic residue N863), and 3) Y836 is not hydrogen bonded to either residue. Each of the three conformers differs from the active state of HNH. The conversion between the inactive and active states involves a local unfolding-refolding process that displaces the Cα and side chain of the catalytic N863 residue by ∼5 Å and ∼10 Å, respectively. In this study, we report the two largest principal components of coordinate variance of the HNH domain throughout molecular dynamics trajectories to establish the interconversion pathways of these conformations. We show that conformation 2 is an obligate step between conformations 1 and 3, which are not directly interconvertible without conformation 2. The loss of hydrogen bonding of the Y836 side chain in conformation 3 likely plays an essential role in activation during local unfolding-refolding of an α-helix containing the catalytic N863. Three single Lys-to-Ala mutants appear to eliminate this substrate-independent activation pathway of the wild-type HNH nuclease, thereby enhancing the fidelity of HNH cleavage.

Common scab disease: structural basis of elicitor recognition in pathogenic Streptomyces species.

In Streptomyces scabiei, the main causative agent of common scab disease of root and tuber crops, the interaction between the substrate-binding protein (SBP) CebE (CebEscab) and cellotriose released by the plant host (KD in the nanomolar range) is the first event for the onset of its pathogenic lifestyle. Here, we report the structure of CebEscab in complex with cellotriose at a resolution of 1.55 Å, adopting a general fold of the B subcluster of SBPs. The interaction between CebEscab and cellotriose involves multiple direct or water-mediated hydrogen bonds and hydrophobic interactions, with the glucose monomer at the non-reducing end occupying the most conserved part of the substrate-binding cleft. As main interactions between the two domains of CebE involve cellotriose itself, the closed conformational state of CebE is performed via an induced-fit ligand binding mechanism where cellotriose binding triggers the domain movement. Analysis of regulon predictions revealed that the signaling pathway from CebE-mediated cellotriose transport to the transcriptional activation of thaxtomin phytotoxin biosynthesis is conserved in Streptomyces spp. causing common scab, except for Streptomyces ipomoeae, which specifically colonizes sweet potatoes and responds to other and yet unknown virulence elicitors. Interestingly, strains belonging to the pathogenic species turgidiscabies and caniscabiei have a cellotriose-binding protein orthologous to the CebE protein of the saprophytic species Streptomyces reticuli with lower affinity for its substrate (KD in the micromolar range), suggesting higher cellotriose concentrations for perception of their host. Our work also provides the structural basis for the uptake of cellobiose and cellotriose by non-pathogenic cellulose-decomposing Streptomyces species.IMPORTANCECommon scab is a disease caused by a few Streptomyces species that affects important root and tuber crops including potato, beet, radish, and parsnip, resulting in major economic losses worldwide. In this work, we unveiled the molecular basis of host recognition by these pathogens by solving the structure of the sugar-binding protein CebE of Streptomyces scabiei in complex with cellotriose, the main elicitor of the pathogenic lifestyle of these bacteria. We further revealed that the signaling pathway from CebE-mediated transport of cellotriose is conserved in all pathogenic species except Streptomyces ipomoeae, which causes soft rot disease in sweet potatoes. Our work also provides the structural basis of the uptake of cellobiose and cellotriose in saprophytic Streptomyces species, the first step activating the expression of the enzymatic system degrading the most abundant polysaccharide on earth, cellulose.