Ligand-bound Forms Research Articles

Clostridium perfringens chitinases, key enzymes during early stages of necrotic enteritis in broiler chickens.

The interaction between bacteria and the intestinal mucus is crucial during the early pathogenesis of many enteric diseases in mammals. A critical step in this process employed by both commensal and pathogenic bacteria focuses on the breakdown of the protective layer presented by the intestinal mucus by mucolytic enzymes. C. perfringens type G, the causative agent of necrotic enteritis in broilers, produces two glycosyl hydrolase family 18 chitinases, ChiA and ChiB, which display distinct substrate preferences. Whereas ChiB preferentially processes linear substrates such as chitin, ChiA prefers larger and more branched substrates, such as carbohydrates presented by the chicken intestinal mucus. Here, we show via crystal structures of ChiA and ChiB in the apo and ligand-bound forms that the two enzymes display structural features that explain their substrate preferences providing a structural blueprint for further interrogation of their function and inhibition. This research focusses on the roles of ChiA and ChiB in bacterial proliferation and mucosal attachment, two processes leading to colonization and invasion of the gut. ChiA and ChiB, either supplemented or produced by the bacteria, led to a significant increase in C. perfringens growth. In addition to nutrient acquisition, the importance of chitinases in bacterial attachment to the mucus layer was shown using an in vitro binding assay of C. perfringens to chicken intestinal mucus. Both an in vivo colonization trial and a necrotic enteritis trial were conducted, demonstrating that a ChiA chitinase mutant strain was less capable to colonize the intestine and was hampered in its disease-causing ability as compared to the wild-type strain. Our findings reveal that the pathogen-specific chitinases produced by C. perfringens type G strains play a fundamental role during colonization, suggesting their potential as vaccine targets.

Structural insights of an LCP protein-LytR-from Streptococcus dysgalactiae subs. dysgalactiae through biophysical and in silico methods.

The rise of antibiotic-resistant bacterial strains has become a critical health concern. According to the World Health Organization, the market introduction of new antibiotics is alarmingly sparse, underscoring the need for novel therapeutic targets. The LytR-CpsA-Psr (LCP) family of proteins, which facilitate the insertion of cell wall glycopolymers (CWGPs) like teichoic acids into peptidoglycan, has emerged as a promising target for antibiotic development. LCP proteins are crucial in bacterial adhesion and biofilm formation, making them attractive for disrupting these processes. This study investigated the structural and functional characteristics of the LCP domain of LytR from Streptococcus dysgalactiae subsp. dysgalactiae. The protein structure was solved by X-ray Crystallography at 2.80Å resolution. Small-angle X-ray scattering (SAXS) data were collected to examine potential conformational differences between the free and ligand-bound forms of the LytR LCP domain. Additionally, docking and molecular dynamics (MD) simulations were used to predict the interactions and conversion of ATP to ADP and AMP. Experimental validation of these predictions was performed using malachite green activity assays. The determined structure of the LCP domain revealed a fold highly similar to those of homologous proteins while SAXS data indicated potential conformational differences between the ligand-free and ligand-bound forms, suggesting a more compact conformation during catalysis, upon ligand binding. Docking and MD simulations predicted that the LytR LCP domain could interact with ADP and ATP and catalyze their conversion to AMP. These predictions were experimentally validated by malachite green activity assays, confirming the protein's functional versatility. The study provides significant insights into the structural features and functional capabilities of the LCP domain of LytR from S. dysgalactiae subsp. dysgalactiae. These findings pave the way for designing targeted therapies against antibiotic-resistant bacteria and offer strategies to disrupt bacterial biofilm formation.

HSADab: A comprehensive database for human serum albumin

Human Serum Albumin (HSA), the most abundant protein in human body fluids, plays a crucial role in the transportation, absorption, metabolism, distribution, and excretion of drugs, significantly influencing their therapeutic efficacy. Despite the importance of HSA as a drug target, the available data on its interactions with external agents, such as drug-like molecules and antibodies, are limited, posing challenges for molecular modeling investigations and the development of empirical scoring functions or machine learning predictors for this target. Furthermore, the reported entries in existing databases often contain major inconsistencies due to varied experiments and conditions, raising concerns about data quality. To address these issues, a pioneering database, HSADab, was established through an extensive review of >30,000 scientific publications published between 1987 and 2023. The database encompasses over 5000 affinity data points at multiple temperatures and >130 crystal structures, including both ligand-bound and apo forms. The current HSADab resource (www.hsadab.cn) serves as a reliable foundation for validating molecular simulation protocols, such as traditional virtual screening workflows using docking, end-point, and al-chemical free energy techniques. Additionally, it provides a valuable data source for the implementation of machine learning predictors, including plasma protein binding models and plasma protein-based drug design models.

Allosteric modulation of fluorescence revealed by hydrogen bond dynamics in a genetically encoded maltose biosensor.

Genetically encoded fluorescent biosensors (GEFBs) proved to be reliable tracers for many metabolites and cellular processes. In the simplest case, a fluorescent protein (FP) is genetically fused to a sensing protein which undergoes a conformational change upon ligand binding. This drives a rearrangement in the chromophore environment and changes the spectral properties of the FP. Structural determinants of successful biosensors are revealed only in hindsight when the crystal structures of both ligand-bound and ligand-free forms are available. This makes the development of new biosensors for desired analytes a long trial-and-error process. In the current study, we conducted μs-long all atom molecular dynamics (MD) simulations of a maltose biosensor in both the apo (dark) and holo (bright) forms. We performed detailed hydrogen bond occupancy analyses to shed light on the mechanism of ligand induced conformational change in the sensor protein and its allosteric effect on the chromophore environment. We find that two strong indicators for distinguishing bright and dark states of biosensors are due to substantial changes in hydrogen bond dynamics in the system and solvent accessibility of the chromophore.

Small angle X-ray scattering analysis of thermophilic cytochrome P450 CYP119 and the effects of the N-terminal histidine tag

Combining size exclusion chromatography-small angle X-ray scattering (SEC-SAXS) and molecular dynamics (MD) analysis is a promising approach to investigate protein behavior in solution, particularly for understanding conformational changes due to substrate binding in cytochrome P450s (CYPs). This study investigates conformational changes in CYP119, a thermophilic CYP from Sulfolobus acidocaldarius that exhibits structural flexibility similar to mammalian CYPs. Although the crystal structure of ligand-free (open state) and ligand-bound (closed state) forms of CYP119 is known, the overall structure of the enzyme in solution has not been explored until now. It was found that theoretical scattering profiles from the crystal structures of CYP119 did not align with the SAXS data, but conformers from MD simulations, particularly starting from the open state (46 % of all frames), agreed well. Interestingly, a small percentage of closed-state conformers also fit the data (9 %), suggesting ligand-free CYP119 samples ligand-bound conformations. Ab initio SAXS models for N-His tagged CYP119 revealed a tail-like unfolded structure impacting protein flexibility, which was confirmed by in silico modeling. SEC-SAXS analysis of N-His CYP119 indicated pentameric structures in addition to monomers in solution, affecting the stability and activity of the enzyme. This study adds insights into the conformational dynamics of CYP119 in solution.

Ligand bound structure of a 6‐hydroxynicotinic acid 3‐monooxygenase provides mechanistic insights

6-Hydroxynicotinic acid 3-monooxygenase (NicC) is a bacterial enzyme involved in the degradation of nicotinic acid. This enzyme is a Class A flavin-dependent monooxygenase that catalyzes a unique decarboxylative hydroxylation. The unliganded structure of this enzyme has previously been reported and studied using steady- and transient-state kinetics to support a comprehensive kinetic mechanism. Here we report the crystal structure of the H47Q NicC variant in both a ligand-bound (solved to 2.17 Å resolution) and unliganded (1.51 Å resolution) form. Interestingly, in the liganded form, H47Q NicC is bound to 2-mercaptopyridine (2-MP), a contaminant present in the commercial stock of 6-mercaptopyridine-3-carboxylic acid(6-MNA), a substrate analogue. 2-MP binds weakly to H47Q NicC and is not a substrate for the enzyme. Based on kinetic and thermodynamic characterization, we have fortuitously captured a catalytically inactive H47Q NicC•2-MP complex in our crystal structure. This complex reveals interesting mechanistic details about the reaction catalyzed by 6-hydroxynicotinic acid 3-monooxygenase.

Structural insights into CodY activation and DNA recognition.

Virulence factors enable pathogenic bacteria to infect host cells, establish infection, and contribute to disease progressions. In Gram-positive pathogens such as Staphylococcus aureus (Sa) and Enterococcus faecalis (Ef), the pleiotropic transcription factor CodY plays a key role in integrating metabolism and virulence factor expression. However, to date, the structural mechanisms of CodY activation and DNA recognition are not understood. Here, we report the crystal structures of CodY from Sa and Ef in their ligand-free form and their ligand-bound form complexed with DNA. Binding of the ligands-branched chain amino acids and GTP-induces conformational changes in the form of helical shifts that propagate to the homodimer interface and reorient the linker helices and DNA binding domains. DNA binding is mediated by a non-canonical recognition mechanism dictated by DNA shape readout. Furthermore, two CodY dimers bind to two overlapping binding sites in a highly cooperative manner facilitated by cross-dimer interactions and minor groove deformation. Our structural and biochemical data explain how CodY can bind a wide range of substrates, a hallmark of many pleiotropic transcription factors. These data contribute to a better understanding of the mechanisms underlying virulence activation in important human pathogens.

Crystal structure of MAB_4123, a putative flavin-dependent monooxygenase from Mycobacterium abscessus.

Numerous bacteria from different phylae can perform desulfurization reactions of organosulfur compounds. In these degradation or detoxification pathways, two-component flavin-dependent monooxygenases that use flavins (FMN or FAD) as a cofactor play important roles as they catalyse the first steps of these metabolic routes. The TdsC or DszC and MsuC proteins belong to this class of enzymes as they process dibenzothiophene (DBT) and methanesulfinate. Elucidation of their X-ray structures in apo, ligand-bound and cofactor-bound forms has provided important molecular insights into their catalytic reaction. Mycobacterial species have also been shown to possess a DBT degradation pathway, but no structural information is available on these two-component flavin-dependent monooxygenases. In this study, the crystal structure of the uncharacterized MAB_4123 protein from the human pathogen Mycobacterium abscessus is presented. The structure solved at high resolution displays high similarity to homologs from Rhodococcus, Paenibacillus and Pseudomonas species. In silico docking approaches suggest that MAB_4123 binds FMN and may use it as a cofactor. Structural analysis strongly suggests that MAB_4123 is a two-component flavin-dependent monooxygenase that could act as a detoxifying enzyme of organosulfur compounds in mycobacteria.

Intermediate-aided allostery mechanism for α-glucosidase by Xanthene-11v as an inhibitor using residue interaction network analysis

Exploring allosteric inhibition and the discovery of new inhibitor binding sites are important studies in protein regulation mechanisms and drug discovery. Structural and network-based analyses of trajectories resulting from molecular dynamics (MD) simulations have been developed to discover protein dynamics, landscape, functions, and allosteric regions. Here, an experimentally suggested non-competitive inhibitor, xanthene-11v, was considered to explore its allosteric inhibition mechanism in α-glucosidase MAL12. Comparative structural and network analyses were applied to eight 250 ns independent MD simulations, four of which were performed in the free state and four of which were performed in ligand-bound forms. Projected two-dimensional free energy landscapes (FEL) were constructed from the probabilistic distribution of conformations along the first two principal components. The post-simulation analyses of the coordinates, side-chain torsion angles, non-covalent interaction networks, network communities, and their centralities were performed on α-glucosidase conformations and the intermediate sub-states. Important communities of residues have been found that connect the allosteric site to the active site. Some of these residues like Thr307, Arg312, TYR344, ILE345, Phe357, Asp406, Val407, Asp408, and Leu436 are the key messengers in the transition pathway between allosteric and active sites. Evaluating the probability distribution of distances between gate residues including Val407 in one community and Phe158, and Pro65 in another community depicted the closure of this gate due to the inhibitor binding. Six macro states of protein were deduced from the topology of FEL and analysis of conformational preference of free and ligand-bound systems to these macro states shows a combination of lock-and-key, conformational selection, and induced fit mechanisms are effective in ligand binding. All these results reveal structural states, allosteric mechanisms, and key players in the inhibition pathway of α-glucosidase by xanthene-11v.

Molecular dynamics of the ERRγ ligand-binding domain bound with agonist and inverse agonist.

Estrogen-related receptor gamma (ERRγ), the latest member of the ERR family, does not have any known reported natural ligands. Although the crystal structures of the apo, agonist-bound, and inverse agonist-bound ligand-binding domain (LBD) of ERRγ have been solved previously, their dynamic behavior has not been studied. Hence, to explore the intrinsic dynamics of the apo and ligand-bound forms of ERRγ, we applied long-range molecular dynamics (MD) simulations to the crystal structures of the apo and ligand-bound forms of the LBD of ERRγ. Using the MD trajectories, we performed hydrogen bond and binding free energy analysis, which suggested that the agonist displayed more hydrogen bonds with ERRγ than the inverse agonist 4-OHT. However, the binding energy of 4-OHT was higher than that of the agonist GSK4716, indicating that hydrophobic interactions are crucial for the binding of the inverse agonist. From principal component analysis, we observed that the AF-2 helix conformation at the C-terminal domain was similar to the initial structures during simulations, indicating that the AF-2 helix conformation is crucial with respect to the agonist or inverse agonist for further functional activity of ERRγ. In addition, we performed residue network analysis to understand intramolecular signal transduction within the protein. The betweenness centrality suggested that few of the amino acids are important for residue signal transduction in apo and ligand-bound forms. The results from this study may assist in designing better therapeutic compounds against ERRγ associated diseases.

Binding mechanism of andrographolide with intramolecular antiparallel G-quadruplexes of therapeutic importance: an in-silico analysis

ABSTRACT G-quadruplexes are non-canonical DNA structures that emerged as important drug targets. Andrographolide is a plant-derived compound with various pharmacological properties. The binding of andrographolide with CREB1 G-quadruplex and Thrombin Binding Aptamer (TBA) was estimated using molecular docking, dynamics, and simulation. The data demonstrated the stabilising effect of the ligand on G-quadruplexes. Andrographolide binds to the CREB1 G-quadruplex by forming hydrogen bonds with the terminal residues and thymine and cytosine residues in the loop region with a binding energy of – 7.30 kcal/mol. At the same time, the TBA G-quadruplex structure formed hydrogen bonds with the residues in the quartet and loop region with binding energy – 6.61 kcal/mol. The Molecular Mechanics Poisson-Boltzmann Surface Area analysis revealed that the contribution from electrostatic energy is higher (−109.21 ± 51.40 KJ/mol) for CREB1 than for TBA (−41.49 ± 27.25 KJ/mol). Principle component analysis illustrated that the eigenvalues are much higher for the unbound forms than for the ligand-bound forms. These findings indicate that andrographolide could potentially target G-quadruplex forming DNA sequences in various oncogenes and can have multiple therapeutic applications.

Insights into receptor structure and dynamics at the surface of living cells

Evaluating protein structures in living cells remains a challenge. Here, we investigate Interleukin-4 receptor alpha (IL-4Rα) into which the non-canonical amino acid bicyclo[6.1.0]nonyne-lysine (BCNK) is incorporated by genetic code expansion. Bioorthogonal click labeling is performed with tetrazine-conjugated dyes. To quantify the reaction yield in situ, we develop brightness-calibrated ratiometric imaging, a protocol where fluorescent signals in confocal multi-color images are ascribed to local concentrations. Screening receptor mutants bearing BCNK in the extracellular domain uncovered site-specific variations of both click efficiency and Interleukin-4 binding affinity, indicating subtle well-defined structural perturbations. Molecular dynamics and continuum electrostatics calculations suggest solvent polarization to determine site-specific variations of BCNK reactivity. Strikingly, signatures of differential click efficiency, measured for IL-4Rα in ligand-bound and free form, mirror sub-angstrom deformations of the protein backbone at corresponding locations. Thus, click efficiency by itself represents a remarkably informative readout linked to protein structure and dynamics in the native plasma membrane.

Molecular insights into the catalytic mechanism of plasticizer degradation by a monoalkyl phthalate hydrolase

Phthalate acid esters (PAEs), a group of xenobiotic compounds used extensively as plasticizers, have attracted increasing concern for adverse effects to human health and the environment. Microbial degradation relying on PAE hydrolases is a promising treatment. However, only a limited number of PAE hydrolases were characterized to date. Here we report the structures of MehpH, a monoalkyl phthalate (MBP) hydrolase that catalyzes the reaction of MBP to phthalic acid and the corresponding alcohol, in apo and ligand-bound form. The structures reveal a positively-charged catalytic center, complementary to the negatively-charged carboxyl group on MBP, and a penetrating tunnel that serves as exit of alcohol. The study provides a first glimpse into the enzyme-substrate binding model for PAE hydrolases, leading strong support to the development of better enzymes in the future.

The binding of nitrogen-donor ligands to the ferric and ferrous forms of cytochrome P450 enzymes

The cytochrome P450 superfamily of heme-thiolate monooxygenase enzymes can catalyse various oxidation reactions. The addition of a substrate or an inhibitor ligand induces changes in the absorption spectrum of these enzymes and UV–visible (UV–vis) absorbance spectroscopy is the most common and readily available technique used to interrogate their heme and active site environment. Nitrogen-containing ligands can inhibit the catalytic cycle of heme enzymes by interacting with the heme. Here we evaluate the binding of imidazole and pyridine-based ligands to the ferric and ferrous forms of a selection of bacterial cytochrome P450 enzymes using UV–visible absorbance spectroscopy. The majority of these ligands interact with the heme as one would expect for type II nitrogen directly coordinated to a ferric heme-thiolate species. However, the spectroscopic changes observed in the ligand-bound ferrous forms indicated differences in the heme environment across these P450 enzyme/ligand combinations. Multiple species were observed in the UV–vis spectra of the ferrous ligand-bound P450s. None of the enzymes gave rise to the isolation of a single species with a Soret band at ∼442–447 nm, indicative of a 6-coordinate ferrous thiolate species with a nitrogen-donor ligand. A ferrous species with Soret band at ∼427 nm coupled with an α-band of increased intensity was observed with the imidazole ligands. With some enzyme-ligand combinations reduction resulted in breaking of the iron‑nitrogen bond yielding a 5-coordinate high-spin ferrous species. In other instances, the ferrous form was readily oxidised back to the ferric form on addition of the ligand.

Structures of the ADGRG2-Gs complex in apo and ligand-bound forms.

Adhesion G protein-coupled receptors are elusive in terms of their structural information and ligands. Here, we solved the cryogenic-electron microscopy (cryo-EM) structure of apo-ADGRG2, an essential membrane receptor for maintaining male fertility, in complex with a Gs trimer. Whereas the formations of two kinks were determinants of the active state, identification of a potential ligand-binding pocket in ADGRG2 facilitated the screening and identification of dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate and deoxycorticosterone as potential ligands of ADGRG2. The cryo-EM structures of DHEA-ADGRG2-Gs provided interaction details for DHEA within the seven transmembrane domains of ADGRG2. Collectively, our data provide a structural basis for the activation and signaling of ADGRG2, as well as characterization of steroid hormones as ADGRG2 ligands, which might be used as useful tools for further functional studies of the orphan ADGRG2.

Design and synthesis of novel benzimidazole derivatives as potential Pseudomonas aeruginosa anti-biofilm agents inhibiting LasR: Evidence from comprehensive molecular dynamics simulation and in vitro investigation

Quorum sensing (QS) inhibition is one of the potential methods to target bacterial infection. In this study, comprehensive molecular dynamics simulation (MDS) experiments were conducted on the LasR structure to understand its structural dynamic behavior either in its ligand-free form or in its ligand-bound form (i.e. agonist or antagonist). The results revealed that LasR structure is significantly unstable in its ligand-free and antagonist-bound forms and such structural instability led eventually to complete dissociation of the functioning LasR dimeric form. Accordingly, twenty-eight benzimidazole derivatives were designed, synthesized as potential LasR antagonists, and characterized in vitro as QS inhibitors. Compounds 3d and 7f disclosed the highest percentage inhibition in biofilm formation, pyocyanin, and rhamnolipids production in Pseudomonas aeruginosa (71.70%, 68.70%, 54.00%) and (68.90%, 68.00%, 51.80%), respectively. MDS experiments revealed that these compounds as inhibitors, particularly, 3d, 7f, 8a, and 9g induce LasR structure instability and complete dissociation of its functioning dimeric form similarly to the previously reported inhibitor bromophenethyl-2-nitrobenzamide (BPNB). Furthermore, gene expression assays as another mechanism targeting quorum sensing genes to prove the inhibitory activity of these compounds on virulence factors, revealed that a number of the synthesized compounds were able to downregulate lasR (e.g. 3d and 7f by 61.70% and 26.00%, respectively) and rhlR (e.g. 7f by 16.30%) expressions. The results presented here provide a functional model for LasR that could guide future design of LasR inhibitors.

Cryo-EM structures define ubiquinone-10 binding to mitochondrial complex I and conformational transitions accompanying Q-site occupancy

Mitochondrial complex I is a central metabolic enzyme that uses the reducing potential of NADH to reduce ubiquinone-10 (Q10) and drive four protons across the inner mitochondrial membrane, powering oxidative phosphorylation. Although many complex I structures are now available, the mechanisms of Q10 reduction and energy transduction remain controversial. Here, we reconstitute mammalian complex I into phospholipid nanodiscs with exogenous Q10. Using cryo-EM, we reveal a Q10 molecule occupying the full length of the Q-binding site in the ‘active’ (ready-to-go) resting state together with a matching substrate-free structure, and apply molecular dynamics simulations to propose how the charge states of key residues influence the Q10 binding pose. By comparing ligand-bound and ligand-free forms of the ‘deactive’ resting state (that require reactivating to catalyse), we begin to define how substrate binding restructures the deactive Q-binding site, providing insights into its physiological and mechanistic relevance.

Native Ambient Mass Spectrometry Imaging of Ligand-Bound and Metal-Bound Proteins in Rat Brain.

Label-free spatial mapping of the noncovalent interactions of proteins in their tissue environment has the potential to revolutionize life sciences research by providing opportunities for the interrogation of disease progression, drug interactions, and structural and molecular biology more broadly. Here, we demonstrate mass spectrometry imaging of endogenous intact noncovalent protein-ligand complexes in rat brain. The spatial distributions of a range of ligand-bound and metal-bound proteins were mapped in thin tissue sections by use of nanospray-desorption electrospray ionization. Proteins were identified directly from the tissue by top-down mass spectrometry. Three GDP-binding proteins (ADP ribosylation factor ARF3, ARF1, and GTPase Ran) were detected, identified, and imaged in their ligand-bound form. The nature of the ligand was confirmed by multiple rounds of tandem mass spectrometry. In addition, the metal-binding proteins parvalbumin-α and carbonic anhydrase 2 were detected, identified, and imaged in their native form, i.e., parvalbumin-α + 2Ca2+ and carbonic anhydrase + Zn2+. GTPase Ran was detected with both GDP and Mg2+ bound. Several natively monomeric proteins displaying distinct spatial distributions were also identified by top-down mass spectrometry. Protein mass spectrometry imaging was achieved at a spatial resolution of 200 μm.

Molecular Basis for the Substrate Promiscuity of Isopentenyl Phosphate Kinase from Candidatus methanomethylophilus alvus.

Isopentenyl phosphate kinases (IPKs) catalyze the ATP-dependent phosphorylation of isopentenyl monophosphate (IP) to isopentenyl diphosphate (IPP) in the alternate mevalonate pathways of the archaea and plant cytoplasm. In recent years, IPKs have also been employed in artificial biosynthetic pathways called "(iso) prenol pathways" that utilize promiscuous kinases to sequentially phosphorylate (iso) prenol and generate the isoprenoid precursors IPP and dimethylallyl diphosphate (DMAPP). Furthermore, IPKs have garnered attention for their impressive substrate promiscuity toward non-natural alkyl-monophosphates (alkyl-Ps), which has prompted their utilization as biocatalysts for the generation of novel isoprenoids. However, none of the IPK crystal structures currently available contain non-natural substrates, leaving the roles of active-site residues in substrate promiscuity ambiguous. To address this, we present herein the high-resolution crystal structures of an IPK from Candidatus methanomethylophilus alvus (CMA) in the apo form and bound to natural and non-natural substrates. Additionally, we describe active-site engineering studies leading to enzyme variants with broadened substrate scope, as well as structure determination of two such variants (Ile74Ala and Ile146Ala) bound to non-natural alkyl-Ps. Collectively, our crystallographic studies compare six structures of CMA variants in different ligand-bound forms and highlight contrasting structural dynamics of the two substrate-binding sites. Furthermore, the structural and mutational studies confirm a novel role of the highly conserved DVTGG motif in catalysis, both in CMA and in IPKs at large. As such, the current study provides a molecular basis for the substrate-binding modes and catalytic performance of CMA toward the goal of developing IPKs into useful biocatalysts.

Structure and mutation analysis of the hexameric P4 from Pseudomonas aeruginosa phage phiYY

phiYY is a foremost member of Cystoviridae isolated from Pseudomonas aeruginosa. Its P4 protein with NTPase activity is a molecular motor for their genome packing during viral particle assembly. Previously studies on the P4 from four Pseudomonas phages phi6, phi8, phi12 and phi13 reveal that despite of belonging to the same protein family, they are unique in sequence, structure and biochemical properties. To better understand the structure and function of phiYY P4, four crystal structures of phiYY P4 in apo-form or combined with different ligands were solved at the resolution between 1.85 Å and 2.43 Å, which showed drastic conformation change of the H1 motif in ligand-bound forms compared with in apo-form, a four residue-mutation at the ligand binding pocket abolished its ATPase activity. Furthermore, the truncation mutation of the 50 residues at the C-terminal did not impair the hexamerization and ATP hydrolysis.