pH-dependent Conformational Change Research Articles

PH-Dependent Conformational Changes Due to Ionizable Residues in a Hydrophobic Protein Interior: The Study of L25K and L125K Variants of SNase.

Ionizable residues in the hydrophobic interior of certain proteins are known to play important roles in life processes like energy transduction and enzyme catalysis. These internal ionizable residues show experimental apparent pKa values having large shifts as compared to their values in solution. In the present work, we study the pH-dependent conformational changes undergone by two variants of staphylococcal nuclease (SNase), L25K and L125K, using pH replica exchange molecular dynamics (pH-REMD) in explicit solvent. Our results show that the observed pKa of Lys25 and Lys125 are significantly different than their pKa in solution. We observed that the internal lysine residues prefer to be water-exposed when protonated at low pH, but they remain buried within the hydrophobic pocket when deprotonated at high pH. Using thermodynamic laws, we estimate the microscopic conformation-specific pKa of the water-exposed and buried conformations of the internal lysine residues and explain their relation to the macroscopic observed pKa values. We present the differences in the microscopic mechanisms that lead to similar experimentally observed apparent pKa of Lys25 and Lys125, and explain the need of thermodynamic models of different complexities to account for our calculations. We see that L25K displays pH-dependent fluctuations throughout the entire β barrel and the α1 helix. In contrast, pH-independent fluctuations are observed in L125K, primarily limited to the α3 helix. The present computational study offers a detailed atomistic understanding of the determinants of the observed anomalous pKa of internal ionizable residues, bolstering the experimental findings.

A study of the endocytosis mechanism and transendothelial activity of lung-targeted GALA-modified liposomes

The GALA peptide (WEAALAEALAEALAEHLAEALAEALEALAA) was originally designed to induce the destabilization of endosomal membranes based on its ability to undergo a pH-dependent conformational change from a random coil to an α-helix. We recently found that liposomes modified with GALA peptide (GALA-LPs) extensively accumulate in lung endothelial cells (ECs) after intravenous injection. However, the uptake mechanism of GALA-LPs and their ability to reach alveolar epithelium was unclear. We report herein that GALA-LPs are internalized into ECs via a clathrin-mediated pathway. Surprisingly, GALA-LPs had the ability to pass lung ECs and reach other cells through transcytosis. GALA-LPs were taken up by >70% of lung ECs, while they also accumulated in ~30% of type I alveolar epithelium (ATI). GALA-modified gold nanoparticles were detected in ECs, in the basement membrane and in other cells such as ATI, type II alveolar epithelium (ATII) and alveolar macrophages. Consistent with this result, a significant gene knockdown was achieved in lung epithelium cells using GALA-LPs encapsulating anti-podoplanin siRNA. This indicates that GALA-LPs can be used as a carrier for delivering macromolecules to parenchymal as well as to endothelial cells in the lung. Although caveolae are commonly linked to the transendothelial transport of proteins and antibodies, our data indicate that clathrin-mediated endocytosis might also participate in the transcytosis process.

PH-Dependent Conformational Changes of KcsA Tetramer and Monomer Probed by Raman Spectroscopy.

KcsA is a tetrameric potassium channel formed out of four identical monomeric subunits used as a standard model for selective potassium transport and pH-dependent gating. Large conformational changes are reported for tetramer and monomer upon gating, and the response of the monomer being controversial with the two major studies partially contradicting each other. KcsA was analyzed as functional tetramers embedded in liposomes and as monomer subunits with confocal Raman microscopy under physiological conditions for the active and the closed channel state, using 532 nm excitation to avoid introducing conformational changes during the measurement. Channel function was confirmed using liposome flux assay. While the classic fingerprint region below 1800 rel. cm−1 in the Raman spectrum of the tetramer was unaffected, the CH-stretching region between 2800 and 3200 rel. cm−1 was found to be strongly affected by the conformation. No pH-dependency was observed in the Raman spectra of the monomer subunits, which closely resembled the Raman spectrum of the tetramer in its active conformation, indicating that the open conformation of the monomer and not the closed conformation as postulated may equal the relaxed state of the molecule.

Structural properties of an engineered outer membrane protein G mutant, OmpG-16SL, investigated with infrared spectroscopy

The structural and functional differences between wild type (WT) outer membrane protein G and its two mutants are investigated with Fourier transform infrared spectroscopy. Both mutants have a long extension to the primary sequence to increase the number of β-strands from 14 (wild type) to 16 in an attempt to enlarge the pore diameter. The comparison among proteins is made in terms of pH-dependent conformational changes and thermal stability. Results show that all proteins respond to pH change but at different degrees. At acidic environment, all proteins contain the same number of residues participated in β-sheet structure. However, at neutral pH, the mutants have less ordered structure compared to WT porin. Thermal stability tests show that mutants differ significantly from WT porin at neutral pH. Although the transition temperature is directly proportional with the amount of β-sheet content, the changes in the pre-transition phase that pave the way to structural breakdown are shown to involve interactions among charged residues by two-dimensional correlation spectroscopy analysis. Results of the analysis show that side chain interactions play an active role under increasing temperature. Both mutants have more unordered secondary structure but they respond to pH change in tertiary structure level. Findings of this study provided deeper insight on the active players in structural stability of the WT porin.Communicated by Ramaswamy H. Sarma

Temperature- and pH-dependent protein conformational changes investigated by terahertz dielectric spectroscopy

Polypeptides and protein drugs have received enormous attention from pharmaceutical industry, medical sector and consumer groups because of a favourable combination of their bioactivity, specificity and overall success rate for the treatment of a variety of diseases. The efficacy and safety of drugs may be degraded due to fluctuating environmental factors, accompanied by changes of the natural conformation of protein. Thus, it is necessary and meaningful to evaluate drug status before use. To that end, the evolving new and pre-existing protein activity/conformation technologies have proven to ensure the safety of drug use consistently. Recently, terahertz time-domain spectroscopy (THz-TDS) has demonstrated suitability for label-free and non-destructive detection of polypeptide and protein conformational changes. In this paper, THz optical parameters of pepsin solutions under different temperatures and with varying pH are measured to demonstrate the feasibility and the considerable potential of THz spectroscopic method in detecting protein drugs. In cases where temperature or pH change is apparent, the THz absorption coefficient, the refractive index, and the dielectric loss tangent change noticeably, independently verified by enzyme activity testing. These findings strongly support the conclusion that THz spectroscopy of pepsin solutions can be used for qualitative analysis to identify the folding or unfolding of protein drugs caused by changes of environmental factors, laying the foundation of a new label-free method for quality control of protein drugs.

PH-Dependent Properties of Ionizable Residues in the Hydrophobic Interior of a Protein

Internal ionizable residues in proteins have shifted apparent pKa values with respect their intrinsic pKa in bulk water. To understand the atomistic mechanism responsible for this shift in pKa, we study the pH-dependent conformational changes in mutants of Staphylococcal Nuclease (SNase) having single internal ionizable residues. We used pH Replica Exchange Molecular Dynamics (pH-REMD) for our calculations. We present thermodynamic models to find the ‘conformation-specific pKa values’ of the internal ionizable residues and explain their association with the observed ‘apparent pKa’. Further, we study the pH-dependent structural and thermodynamic properties of ionizable residue pairs within the hydrophobic interior of SNase.

PH-Driven Conformational Reorganization of Proteins: NMR Spectroscopy Study with Buried Lys Residues

The pH sensitivity of proteins is essential for biological energy transduction and for cellular and physiological homeostasis. The thermodynamic basis of pH effects in proteins is well understood but detailed mechanistic understanding of how changes in pH drive conformational reorganization in proteins is not. This was addressed systematically using NMR spectroscopy to characterize pH-dependent conformational changes coupled to the ionization of residues buried in the hydrophobic core of a protein. The study used 25 variants of staphylococcal nuclease with buried Lys residues. The pKa values of 19 of 25 of these internal Lys residues are depressed relative to the normal pKa of Lys in water, some by as much as 5 pH units. This shift in pKa ensures that the ionizable moiety stays neutral at pH values below 10 when it is buried, consistent with what is expected from the unfavorable transfer of a charge from water to a hydrophobic environment. In most cases, ionization of the buried Lys shifts the conformation of the protein into a state where the charged Lys can contact water. This study contributes a detailed description of the location, amplitude and time scale of reorganization coupled to the ionization of buried Lys residues. The structural details of the conformational response were characterized with backbone chemical shift perturbation analysis, titrations of the buried Nζ atom, and in some cases even X-ray crystallography. At least in this protein, the anomalous pKa values of buried groups are governed by the propensity of the protein to reorganize. These results demonstrate that computational modeling of pH dependent processes in proteins will require prediction of alternative conformational states and accurate calculation of free energies; this remains a significant challenge even under the most favorable circumstances.

Subunit-subunit interactions play a key role in the heme-degradation reaction of HutZ from Vibrio cholerae.

HutZ, a dimeric protein, from Vibrio cholerae is a protein that catalyzes the oxygen-dependent degradation of heme. Interestingly, the ascorbic acid-supported heme-degradation activity of HutZ depends on pH: less than 10% of heme is degraded by HutZ at pH 8.0, but nearly 90% of heme is degraded at pH 6.0. We examined here pH-dependent conformational changes in HutZ using fluorescence spectroscopy. Trp109 is estimated to be located approximately 21 Å from heme and is present in a different subunit containing a heme axial ligand. Thus, we postulated that the distance between heme and Trp109 reflects subunit-subunit orientational changes. On the basis of resonance energy transfer from Trp109 to heme, we estimated the distance between heme and Trp109 to be approximately 17 Å at pH 8.0, while the distance increased by less than 2 Å at pH 6.0. We presumed that such changes led to a decrease in electron donation from the proximal histidine, resulting in enhancement of the heme-degradation activity. To confirm this scenario, we mutated Ala31, located at the dimer interface, to valine to alter the distance through the subunit-subunit interaction. The distance between heme and Trp109 for the A31V mutant was elongated to 24-27 Å. Although resonance Raman spectra and reduction rate of heme suggested that this mutation resulted in diminished electron donation from the heme axial ligand, ascorbic acid-supported heme-degradation activity was not observed. Based on our findings, it can be proposed that the relative positioning of two protomers is important in determining the heme degradation rate by HutZ.

Total Synthesis of Emmyguyacins A and B, Potential Fusion Inhibitors of Influenza Virus.

Fungal glycolipids emmyguyacins A and B inhibit the pH-dependent conformational change of hemaglutinin A during replication of the Influenza virus. Herein, we report the first total synthesis and structure confirmation of emmyguyacins A and B. Our efficient route, which involves regioselective functionalization of trehalose, allows rapid access to adequate amounts of chemically pure emmyguyacin analogues including the desoxylate derivatives for SAR studies.

PH-dependent conformational changes of β-amylase/glucose complex crystal measured at room temperature

pH-dependent conformational changes of β-amylase/glucose complex crystal measured at room temperature

Stimuli-triggered reversible switching mechanism between H- and J-type supramolecular assemblies of cationic porphyrins adsorbed on tungsten(VI) oxide surface

Supramolecular organic dye–inorganic semiconductor nanocrystal assemblies are potentially useful in a broad range of technologies and applications, including photovoltaic systems, but the molecular basis of the adsorption of dye molecules onto the semiconductor surfaces remains poorly understood. Herein, we investigated the pH-dependent adsorption and conformational change of two cationic porphyrin stereoisomers [5,10-diphenyl-15,20-di([Formula: see text]-methyl-4-pyridyl)porphyrin (cis-DMPyP) and 5,15-diphenyl-10,20-di([Formula: see text]-methyl-4-pyridyl)porphyrin (trans-DMPyP)] on the tungsten(VI) oxide (WO[Formula: see text] colloid nanoparticle in aqueous media by means of UV-vis absorption spectroscopy. In accordance with the combination of a modified Langmuir adsorption model and Kasha’s exciton coupling model, the molecular orientation and stacking arrangement of DMPyP derivatives on the WO[Formula: see text] colloid surface are discussed in detail. In the trans-DMPyP/WO[Formula: see text] aqueous system, trans-DMPyP molecules adopted flat-on orientation with respect to the WO[Formula: see text] colloid surface and eventually formed head-to-tail [Formula: see text]-dimers regardless of pH conditions. cis-DMPyP molecules in the acidic system also lay flat-on and mainly formed [Formula: see text]-dimers on the WO[Formula: see text] colloid surface, whereas ones in the neutral system exhibited a dominant edge-on orientation and had a higher tendency to form face-to-face [Formula: see text]-dimers. Additionally, we have also convincingly demonstrated the pH-triggered switchable [Formula: see text]-stacking geometry of cis-DMPyP molecules from [Formula: see text]- to [Formula: see text]-dimer and vice versa on the WO[Formula: see text] colloid surface. Such findings will undoubtedly provide a pertinent guideline for the rational design of stimuli-responsive organic-inorganic materials.

(Keynote) Strategical Design and Applications of Responsive Gels with Dynamic Crosslinks

Stimuli-responsive gels are useful soft materials for developing sensors, drug delivery systems and cell cultures. We proposed a novel strategy for designing biomolecularly stimuli-responsive gels that undergo changes in the volume in response to a target biomolecule. Our strategy uses molecular complexes as dynamic crosslinks that dissociate and associate in the presence and absence of a target biomolecule [1]. Using the strategy, we have prepared a variety of responsive gels that exhibit swelling/shrinking behaviors or sol-gel phase transition in response to a target biomolecule. These studies demonstrated that dynamic crosslinks are useful tools for designing stimuli-responsive gels. This paper focuses on strategical design of stimuli-responsive gels using dynamic crosslinks and their applications to drug delivery, sensors and cell cultures. Biomolecularly stimuli-responsive gels that can swell in response to a target biomolecule have been designed as biomolecule-crosslinked gels using biomolecular complex crosslinks such as antigen-antibody complexes and DNA duplexes [2]. As biomolecularly stimuli-responsive gels that can shrink in the presence of a target biomolecule, we have prepared biomolecule-imprinted gels by molecular imprinting that uses cyclodextrin (CD), lectin, antibody and DNA as ligands [3, 4]. In addition, polypeptides with inherent secondary structures such as α-helix and random coil were utilized for designing molecularly imprinted gels with molecular recognition sites [5]. The binding capacity of molecularly imprinted polypeptides gels was regulated by pH-dependent conformational changes. The fascinating properties of the molecularly imprinted polypeptide gels suggest that they exhibit potential as smart drug carriers. We prepared micro- and nano-sized molecularly stimuli-responsive gels such as particles, thin films and microvalves. For example, molecularly imprinted gel layers with molecular recognition sites were prepared on surface plasmon resonance (SPR) sensor chips by surface-initiated atom transfer radical polymerization (SI-ATRP) [6]. The molecularly imprinted gel layer chips exhibited a large SPR signal and binding constant, compared to nonimprinted gel layer chips. To fabricate self-regulated micro-total analysis systems (μ-TAS), we prepared various micro-sized gels that exhibited quick swelling or shrinkage in response to a target molecule in a microchannel by photopolymerization using a fluorescence microscope [7]. The flow rate of a microchannel was autonomously regulated by the molecularly stimuli-responsive swelling or shrinking of their micro-gels as smart microvalves. Biotin-conjugated four-armed poly(ethylene glycol) (biotinylated Tetra-PEG) was designed as molecularly stimuli-responsive sol–gel transition polymers that underwent the phase transition from a sol to a gel state in response to avidin as a target molecule [8]. When avidin was added to a buffer solution containing biotinylated Tetra-PEG, the solution transformed to a gel state immediately. The addition of free biotin to the resulting gel induced its dissociation to a sol state. In addition, to design DDS reservoirs and cell culture scaffolds, we designed dual stimuli-responsive sol–gel transition polymers that responded to photo/temperature- and photo/molecule-stimuli. References Miyata, T. Polym J. 2010, 42, 277–289.Miyata, T.; Asami, N.; Uragami, T. Nature 1999, 399, 766–769.Miyata, T.; Jige, M.; Nakaminami, T.; Uragami, T. PNAS 2006; 103: 1190–1193.Kawamura, A.; Kiguchi, T.; Nishihata, T.; Uragami, T.; Miyata, T. Chem. Commun. 2014, 50, 11101–11103.Matsumoto, K.; Kawamura, A.; Miyata, T. Macromolecules 2017, 50, 2136–2144.Kuriu, Y.; Kawamura, A.; Uragami, T.; Miyata, T. Chem. Lett. 2014, 43, 825–827.Shiraki, Y.; Tsuruta, K.; Morimoto, J.; Ohba, C.; Kawamura, A.; Yoshida, R.; Kawano, R.; Uragami, T.; Miyata, T. Macromol. Rapid. Commun. 2015, 36, 515–519.Norioka, C.; Okita, K.; Mukada, M.; Kawamura, A.; Miyata, T. Polym. Chem. 2017, 8, 6378–6385.

Demystifying the pH dependent conformational changes of human heparanase pertaining to structure-function relationships: an in silico approach.

Heparanase (HPSE) is an endo-β-D-glucuronidase that has diverse functions in mammals which includes cell survival, cell adhesion and cell migration. HPSE features both enzymatic and non-enzymatic functionalities in a pH dependent manner. Hence, in this study, an extensive molecular dynamics simulation, molecular docking, protein Angular dispersion analysis were performed for apo form and holo forms to understand its conformational changes at varied pH conditions. On comparative conformational analysis of apo and holo forms, it was inferred that the HSPE has undergone pH dependent structural changes, thereby affecting the binding of Heparan sulfate proteoglycan (HSPG). Moreover, HPSE also showed favourable structural changes for optimal binding of HSPG at pH 5.0 and 6.0, as inferred from functional flap displacements within HPSE. Thus, this study provides significant insights on optimal pH for HPSE to exhibit its enzymatic activity. The outcome of this study shall aid in ideal lead generation for targeting HPSE mediated disease conditions.

Biophysical, photochemical and biochemical characterization of a protease from Aspergillus tamarii URM4634

Circular dichroism (CD) and fluorescence spectroscopy (FS) were used to monitor the pH-dependent conformational and structural stability changes induced by temperature and UV light on the protease from Aspergillus tamarii URM4634 at different pH values. The formation of photoproducts, such as N–formylkynurenine, dityrosine and kynurenine, were monitored with FS. The pH-dependent melting temperatures (Tm) were determined using CD and FS from 20 to 90 °C. Conformational changes were correlated with the pH-dependent biochemical activities. CD revealed that the protease is rich in α-helices. Thermal denaturation was irreversible at all pH range and displayed Tm values from 42.8 to 67.8 °C (CD) and from 38 to 60.3 °C (FS), which the highest Tm was observed at pH 6. The light and temperature induced to the formation of photoproducts was more intense at high pH value. Despite the biochemical data shows optimum pH 9, the highest stability was at pH 6, maintaining 100% of activity after 24 h. The acquired data permits to select the best physicochemical parameters to secure the optimal activity and stability when used in biotechnological applications. Furthermore, the conformal changes induced by temperature in the protein are directly correlated with its level of biochemical activity.

PH-Dependent Conformations for Hyperbranched Poly(ethylenimine) from All-Atom Molecular Dynamics

Hyperbranched poly(ethylene imine) (PEI) with its high amino content is a versatile functional polymer that is widely used as a gene delivery vector in biological applications and as a ligand and precursor for ion exchange resins and membranes in water purification and metal recovery. We report here the first fully atomistic model of a 25 kDa hyperbranched PEI macromolecule. We utilized this model to carry out molecular dynamics (MD) simulations with explicit water molecules and chloride ions to study pH-dependent conformational changes. We find that growing the PEI macromolecule sequentially from the monomers yields atomistic structures whose sizes (radius of gyration) are in good agreement with the small angle neutron scattering experiments. Our analysis of the structural properties from MD simulations shows that conformations of the hyperbranched PEI exhibit oblate ellipsoidal elongation at low pH compared to high or neutral pH but with no significant changes in the corresponding sizes. This fully atom...

Structural basis for the role of serine-rich repeat proteins from Lactobacillus reuteri in gut microbe–host interactions

Lactobacillus reuteri, a Gram-positive bacterial species inhabiting the gastrointestinal tract of vertebrates, displays remarkable host adaptation. Previous mutational analyses of rodent strain L. reuteri 100-23C identified a gene encoding a predicted surface-exposed serine-rich repeat protein (SRRP100-23) that was vital for L. reuteri biofilm formation in mice. SRRPs have emerged as an important group of surface proteins on many pathogens, but no structural information is available in commensal bacteria. Here we report the 2.00-Å and 1.92-Å crystal structures of the binding regions (BRs) of SRRP100-23 and SRRP53608 from L. reuteri ATCC 53608, revealing a unique β-solenoid fold in this important adhesin family. SRRP53608-BR bound to host epithelial cells and DNA at neutral pH and recognized polygalacturonic acid (PGA), rhamnogalacturonan I, or chondroitin sulfate A at acidic pH. Mutagenesis confirmed the role of the BR putative binding site in the interaction of SRRP53608-BR with PGA. Long molecular dynamics simulations showed that SRRP53608-BR undergoes a pH-dependent conformational change. Together, these findings provide mechanistic insights into the role of SRRPs in host-microbe interactions and open avenues of research into the use of biofilm-forming probiotics against clinically important pathogens.

Structural Insights into the pH-Dependent Conformational Change and Collagen Recognition of the Human Mannose Receptor

Mannose receptor (MR, CD206) is an endocytic receptor on microphages and dendritic cells. It recognizes multiple ligands and plays important roles inregulating immune responses and maintaining glycoprotein homeostasis. However, the structure and functional mechanism of MR remain unclear. Here we determine the crystal structures of the N-terminal fragments of MR and reveal the potential binding mode of collagen on the fibronectin II domain. The SAXS and other biophysical data suggest that MR adopts an extended conformation at physiological pH and undergoes conformational changes as pH decreases, resulting in a compact conformation in an acidic environment. Moreover, biochemical data show that MR binds to collagen in a Ca2+-enhanced manner at physiological pH, whereas Ca2+ has no effect on the binding at acidic pH. These results provide a model for the dynamic mechanism of MR regarding its ligand binding and release during the recycling between cell surface and endosomes.

Structure of Human M-type Phospholipase A2 Receptor Revealed by Cryo-Electron Microscopy

M-type phospholipase A2 receptor (M-PLA2R) is a member of the mannose receptor family and known as the receptor of secretory phospholipase A2s. It has also been identified as the major autoantigen of idiopathic membranous nephropathy, one of the most common causes for nephrotic syndrome in adults. Here we determine the structure of human M-PLA2R ectodomain by cryo-electron microscopy. The results show that the ectodomain has high internal flexibility and forms a compact dual-ring-shaped conformation at acidic pH and adopts extended conformations at basic pH. The inter-domain interactions of human M-PLA2R are explored by the binding studies with individual domains, showing the mechanism of the conformational change. In addition, the biochemical data suggest that mouse M-PLA2R recognizes mouse secretory phospholipase A2-G1B only at physiological or basic pH, rather than at acidic pH. These results suggest that the pH-dependent conformational change might play important roles in the functional activities of M-PLA2R such as ligand binding and release, and may also be relevant to the immunogenicity in membranous nephropathy.

A Helicobacter pylori Vacuolating Cytotoxin A: Mouse DHFR Fusion Protein Triggers Dye Release from Liposomes

The membrane perturbing action of the VacA toxin from Helicobacter pylori is responsible for vacuole formation in intracellular compartments and the induction of apoptosis. The VacA toxin contains 2 major domains, p33 and p55, which are involved in receptor binding and membrane pore formation, respectively. Improved methodologies for VacA purification and assays are urgently needed for further detailed investigations on the mechanism of action of this significant virulence factor. We found that by fusing mouse DHFR with the N-terminus of the full-length (p88) VacA toxin, expression levels in recombinant E. coli were substantially increased when compared to the conventional (His)6-tagged protein. The DHFR-VacA fusion protein was active in sulforhodamine dye-release assays using liposomes at acidic pH in a concentration-dependent manner. Enzymatic activity of DHFR in the fusion protein was comparable to a commercial reference sample of purified DHFR; however, activity was insensitive to inhibition by methotrexate. Our findings suggest that the VacA p88 toxin with a modified N-terminus still maintains its capability for membrane insertion and that pH-dependent conformational changes occur during interaction of VacA with membranes.

Structural and dynamic insights into the role of conformational switching in the nuclease activity of the Xanthomonas albilineans Cas2 in CRISPR-mediated adaptive immunity.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins constitute a microbial, adaptive immune system countering invading nucleic acids. Cas2 is a universal Cas protein found in all types of CRISPR-Cas systems, and its role is implicated in new spacer acquisition into CRISPR loci. In subtype I-C CRISPR-Cas systems, Cas2 proteins are metal-dependent double-stranded DNA (dsDNA) nucleases, and a pH-dependent conformational transition has been proposed as a prerequisite for catalytic action. Here, we report the crystal structure of Xanthomonas albilineans Cas2 (XaCas2) and provide experimental evidence of a pH-dependent conformational change during functional activation. XaCas2 crystallized at an acidic pH represented a catalytically inactive conformational state in which two Asp8 residues were too far apart to coordinate a single catalytic metal ion. Consistently, XaCas2 exhibited dsDNA nuclease activity only under neutral and basic conditions. Despite the overall structural similarity of the two protomers, significant conformational heterogeneity was evident in the putative hinge regions, suggesting that XaCas2 engages in hinge-bending conformational switching. The presence of a Trp residue in the hinge region enabled the investigation of hinge dynamics by fluorescence spectroscopy. The pH dependence of the fluorescence intensity overlapped precisely with that of nuclease activity. Mutational analyses further suggested that conformational activation proceeded via a rigid-body hinge-bending motion as both D8E and hinge mutations significantly reduced nuclease activity. Together, our results reveal strong correlations between the conformational states, catalytic activity, and hinge dynamics of XaCas2, and provide structural and dynamic insights into the conformational activation of the nuclease function of Cas2.