A conductive catecholate-based framework coordinated with unsaturated bismuth boosts CO2 electroreduction to formate.

Bismuth-based metal-organic frameworks (Bi-MOFs) have received attention in electrochemical CO2-to-formate conversion. However, the low conductivity and saturated coordination of Bi-MOFs usually lead to poor performance, which severely limits their widespread application. Herein, a conductive catecholate-based framework with Bi-enriched sites (HHTP, 2,3,6,7,10,11-hexahydroxytriphenylene) is constructed and the zigzagging corrugated topology of Bi-HHTP is first unraveled via single-crystal X-ray diffraction. Bi-HHTP possesses excellent electrical conductivity (1.65 S m-1) and unsaturated coordination Bi sites are confirmed by electron paramagnetic resonance spectroscopy. Bi-HHTP exhibited an outstanding performance for selective formate production of 95% with a maximum turnover frequency of 576 h-1 in a flow cell, which surpassed most of the previously reported Bi-MOFs. Significantly, the structure of Bi-HHTP could be well maintained after catalysis. In situ attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) confirms that the key intermediate is *COOH species. Density functional theory (DFT) calculations reveal that the rate-determining step is *COOH species generation, which is consistent with the in situ ATR-FTIR results. DFT calculations confirmed that the unsaturated coordination Bi sites acted as active sites for electrochemical CO2-to-formate conversion. This work provides new insights into the rational design of conductive, stable, and active Bi-MOFs to improve their performance towards electrochemical CO2 reduction.

Allosteric regulation of Senecavirus A 3Cpro proteolytic activity by an endogenous phospholipid.

Seneca virus A (SVA) is an emerging novel picornavirus that has recently been identified as the causative agent of many cases of porcine vesicular diseases in multiple countries. In addition to cleavage of viral polyprotein, the viral 3C protease (3Cpro) plays an important role in the regulation of several physiological processes involved in cellular antiviral responses by cleaving critical cellular proteins. Through a combination of crystallography, untargeted lipidomics, and immunoblotting, we identified the association of SVA 3Cpro with an endogenous phospholipid molecule, which binds to a unique region neighboring the proteolytic site of SVA 3Cpro. Our lipid-binding assays showed that SVA 3Cpro displayed preferred binding to cardiolipin (CL), followed by phosphoinositol-4-phosphate (PI4P) and sulfatide. Importantly, we found that the proteolytic activity of SVA 3Cpro was activated in the presence of the phospholipid, and the enzymatic activity is inhibited when the phospholipid-binding capacity decreased. Interestingly, in the wild-type SVA 3Cpro-substrate peptide structure, the cleavage residue cannot form a covalent binding to the catalytic cysteine residue to form the acyl-enzyme intermediate observed in several picornaviral 3Cpro structures. We observed a decrease in infectivity titers of SVA mutants harboring mutations that impaired the lipid-binding ability of 3Cpro, indicating a positive regulation of SVA infection capacity mediated by phospholipids. Our findings reveal a mutual regulation between the proteolytic activity and phospholipid-binding capacity in SVA 3Cpro, suggesting that endogenous phospholipid may function as an allosteric activator that regulate the enzyme's proteolytic activity during infection.

Exploring AlphaFold2's Performance on Predicting Amino Acid Side-Chain Conformations and Its Utility in Crystal Structure Determination of B318L Protein.

Recent technological breakthroughs in machine-learning-based AlphaFold2 (AF2) are pushing the prediction accuracy of protein structures to an unprecedented level that is on par with experimental structural quality. Despite its outstanding structural modeling capability, further experimental validations and performance assessments of AF2 predictions are still required, thus necessitating the development of integrative structural biology in synergy with both computational and experimental methods. Focusing on the B318L protein that plays an essential role in the African swine fever virus (ASFV) for viral replication, we experimentally demonstrate the high quality of the AF2 predicted model and its practical utility in crystal structural determination. Structural alignment implies that the AF2 model shares nearly the same atomic arrangement as the B318L crystal structure except for some flexible and disordered regions. More importantly, side-chain-based analysis at the individual residue level reveals that AF2's performance is likely dependent on the specific amino acid type and that hydrophobic residues tend to be more accurately predicted by AF2 than hydrophilic residues. Quantitative per-residue RMSD comparisons and further molecular replacement trials suggest that AF2 has a large potential to outperform other computational modeling methods in terms of structural determination. Additionally, it is numerically confirmed that the AF2 model is accurate enough so that it may well potentially withstand experimental data quality to a large extent for structural determination. Finally, an overall structural analysis and molecular docking simulation of the B318L protein are performed. Taken together, our study not only provides new insights into AF2's performance in predicting side-chain conformations but also sheds light upon the significance of AF2 in promoting crystal structural determination, especially when the experimental data quality of the protein crystal is poor.

Structural insights into PA3488-mediated inactivation of Pseudomonas aeruginosa PldA.

PldA, a phospholipase D (PLD) effector, catalyzes hydrolysis of the phosphodiester bonds of glycerophospholipids-the main component of cell membranes-and assists the invasion of the opportunistic pathogen Pseudomonas aeruginosa. As a cognate immunity protein, PA3488 can inhibit the activity of PldA to avoid self-toxicity. However, the precise inhibitory mechanism remains elusive. We determine the crystal structures of full-length and truncated PldA and the cryogenic electron microscopy structure of the PldA-PA3488 complex. Structural analysis reveals that there are different intermediates of PldA between the "open" and "closed" states of the catalytic pocket, accompanied by significant conformational changes in the "lid" region and the peripheral helical domain. Through structure-based mutational analysis, we identify the key residues responsible for the enzymatic activity of PldA. Together, these data provide an insight into the molecular mechanisms of PldA invasion and its neutralization by PA3488, aiding future design of PLD-targeted inhibitors and drugs.

Cu-Based Organic-Inorganic Composite Materials for Electrochemical CO2 Reduction.

Electrochemical CO2 reduction reaction (CO2 RR) is an attractive pathway to convert CO2 into value-added chemicals and fuels. Copper (Cu) is the most effective monometallic catalyst for converting CO2 into multi-carbon products, but suffers from high overpotentials and poor selectivity. Therefore, it is essential to design efficient Cu-based catalyst to improve the selectivity of specific products. Due to the combination of advantages of organic and inorganic composite materials, organic-inorganic composites exhibit high catalytic performance towards CO2 RR, and have been extensively studied. In this review, the research advances of various Cu-based organic-inorganic composite materials in CO2 RR, i. e., organic molecular modified-metal Cu composites, Cu-based molecular catalyst/carbon carrier composites, Cu-based metal organic framework (MOF) composites, and Cu-based covalent organic framework (COF) composites are systematically summarized. Particularly, the synthesis strategies of Cu-based composites, structure-performance relationship, and catalytic mechanisms are discussed. Finally, the opportunities and challenges of Cu-based organic-inorganic composite materials in CO2 RR are proposed.

Insights into the Neutralization and DNA Binding of Toxin-Antitoxin System ParESO-CopASO by Structure-Function Studies.

ParESO-CopASO is a new type II toxin-antitoxin (TA) system in prophage CP4So that plays an essential role in circular CP4So maintenance after the excision in Shewanella oneidensis. The toxin ParESO severely inhibits cell growth, while CopASO functions as an antitoxin to neutralize ParESO toxicity through direct interactions. However, the molecular mechanism of the neutralization and autoregulation of the TA operon transcription remains elusive. In this study, we determined the crystal structure of a ParESO-CopASO complex that adopted an open V-shaped heterotetramer with the organization of ParESO-(CopASO)2-ParESO. The structure showed that upon ParESO binding, the intrinsically disordered C-terminal domain of CopASO was induced to fold into a partially ordered conformation that bound into a positively charged and hydrophobic groove of ParESO. Thermodynamics analysis showed the DNA-binding affinity of CopASO was remarkably higher than that of the purified TA complex, accompanied by the enthalpy change reversion from an exothermic reaction to an endothermic reaction. These results suggested ParESO acts as a de-repressor of the TA operon transcription at the toxin:antitoxin level of 1:1. Site-directed mutagenesis of ParESO identified His91 as the essential residue for its toxicity by cell toxicity assays. Our structure-function studies therefore elucidated the transcriptional regulation mechanism of the ParESO-CopASO pair, and may help to understand the regulation of CP4So maintenance in S. oneidensis.

NUDIM: A non-uniform fast Fourier transform based dual-space constraint iterative reconstruction method in biological electron tomography.

Electron tomography, a powerful imaging tool for studying 3D structures of macromolecular assemblies, always suffers from imperfect reconstruction with limited resolution due to the intrinsic low signal-to-noise ratio (SNR) and inaccessibility to certain tilt angles induced by radiation damage or mechanical limitation. In order to compensate for such insufficient data with low SNR and further improve imaging resolution, prior knowledge constraints about the objects in both real space and reciprocal space are thus exploited during tomographic reconstruction. However, direct Fast Fourier transform (FFT) between real space and reciprocal space remains extraordinarily challenging owing to their inconsistent grid sampling modes, e.g. regular and uniform grid sampling in real space whereas radial or polar grid sampling in reciprocal space. In order to solve such problem, a technique of non-uniform fast Fourier transform (NFFT) has been developed to transform efficiently between non-uniformly sampled grids in real and reciprocal space with sufficient accuracy. In this work, a Non-Uniform fast Fourier transform based Dual-space constraint Iterative reconstruction Method (NUDIM) applicable to biological electron tomography is proposed with a combination of basic concepts from equally sloped tomography (EST) and NFFT based reconstruction. In NUDIM, the use of NFFT can circumvent such grid sampling inconsistency and thus alleviate the stringent equally-sloped sampling requirement in EST reconstruction, while the dual-space constraint iterative procedure can dramatically enhance reconstruction quality. In comparison with conventional reconstruction methods, NUDIM is numerically and experimentally demonstrated to produce superior reconstruction quality with higher contrast, less noise and reduced missing wedge artifacts. More importantly, it is also capable of retrieving part of missing information from a limited number of projections.

Structure and Biochemical Characteristic of the Methyltransferase (MTase) Domain of RNA Capping Enzyme from African Swine Fever Virus.

African swine fever virus (ASFV) is a complex nucleocytoplasmic large DNA virus (NCLDV) that causes a devastating swine disease and it is urgently needed to develop effective anti-ASFV vaccines and drugs. The process of mRNA 5'-end capping is a common characteristic in eukaryotes and many viruses, and the cap structure is required for mRNA stability and efficient translation. The ASFV protein pNP868R was found to have guanylyltransferase (GTase) activity involved in mRNA capping. Here we report the crystal structure of pNP868R methyltransferase (MTase) domain (referred as pNP868RMT) in complex with S-adenosyl-L-methionine (AdoMet). The structure shows the characteristic core fold of the class I MTase family and the AdoMet is bound in a negative-deep groove. Remarkably, the N-terminal extension of pNP868RMT is ordered and keeps away from the AdoMet-binding site, distinct from the close conformation over the active site of poxvirus RNA capping D1 subunit or the largely disordered conformation in most cellular RNA capping MTases. Structure-based mutagenesis studies based on the pNP868RMT-cap analog complex model revealed essential residues involved in substrate recognition and binding. Functional studies suggest the N-terminal extension may play an essential role in substrate recognition instead of AdoMet-binding. A positively charged path stretching from the N-terminal extension to the region around the active site was suggested to provide a favorable electrostatic environment for the binding and approaching of substrate RNA into the active site. Our structure and biochemical studies provide novel insights into the methyltransfer process of mRNA cap catalyzed by pNP868R.IMPORTANCE African swine fever (ASF) is a highly contagious hemorrhagic viral disease in pigs that is caused by African swine fever virus (ASFV). There are no effective drugs or vaccines for protection against ASFV infection till now. The protein pNP868R was predicted to be responsible for process of mRNA 5'-end capping in ASFV, which is essential for mRNA stability and efficient translation. Here, we solved the high-resolution crystal structure of the methyltransferase (MTase) domain of pNP868R. The MTase domain structure shows a canonical class I MTase family fold and the AdoMet binds into a negative pocket. Structure-based mutagenesis studies revealed critical and conserved residues involved in AdoMet-binding and substrate RNA-binding. Notably, both the conformation and the role in MTase activities of the N-terminal extension are distinct from those of previously characterized poxvirus MTase domain. Our structure-function studies provide the basis for potential anti-ASFV inhibitor design targeting the critical enzyme.

Structural and functional characterization of the deep-sea thermophilic bacteriophage GVE2 tailspike protein.

The genome of the thermophilic bacteriophage GVE2 encodes a putative tailspike protein (GVE2 TSP). Here we report the crystal structure of the truncated GVE2 TSP at 2.0-Å resolution lacking 204 amino acid residues at its N-terminus (ΔnGVE2 TSP), possessing a "vase" outline similar to other TSP's structures. However, ΔnGVE2 TSP displays structural characteristics distinct from other TSPs. Despite lacking 204 amino acid residues, the head domain forms an asymmetric trimer compared to symmetric in other TSPs, suggesting that its long N-terminus may be unique to the long-tailed bacteriophages. Furthermore, the α-helix of the neck is 5-7 amino acids longer than that of other TSPs. The most striking feature is that its binding domain consists of a ß-helix with 10 turns, whereas other TSPs have 13 turns, even including the phage Sf6 TSP, which is the closest homologue of GVE2 TSP. The C-terminal structure is also quite different with those of other TSPs. Furthermore, we observed that ΔnGVE2 TSP can slow down growth of its host, demonstrating that this TSP is essential for the phage GVE2 to infect its host. Overall, the structural characteristics suggest that GVE2 TSP may be more primitive than other phage TSPs.

Crystal structure of the nucleoid-associated protein Fis (PA4853) from Pseudomonas aeruginosa.

Factor for inversion stimulation (Fis) is a versatile bacterial nucleoid-associated protein that can directly bind and bend DNA to influence DNA topology. It also plays crucial roles in regulating bacterial virulence factors and in optimizing bacterial adaptation to various environments. Fis from Pseudomonas aeruginosa (PA4853, referred to as PaFis) has recently been found to be required for virulence by regulating the expression of type III secretion system (T3SS) genes. PaFis can specifically bind to the promoter region of exsA, which functions as a T3SS master regulator, to regulate its expression and plays an essential role in transcription elongation from exsB to exsA. Here, the crystal structure of PaFis, which is composed of a four-helix bundle and forms a homodimer, is reported. PaFis shows remarkable structural similarities to the well studied Escherichia coli Fis (EcFis), including an N-terminal flexible loop and a C-terminal helix-turn-helix (HTH) motif. However, the critical residues for Hin-catalyzed DNA inversion in the N-terminal loop of EcFis are not conserved in PaFis and further studies are required to investigate its exact role. A gel-electrophoresis mobility-shift assay showed that PaFis can efficiently bind to the promoter region of exsA. Structure-based mutagenesis revealed that several conserved basic residues in the HTH motif play essential roles in DNA binding. These structural and biochemical studies may help in understanding the role of PaFis in the regulation of T3SS expression and in virulence.

Characterization of the Pseudomonas aeruginosa T6SS PldB immunity proteins PA5086, PA5087 and PA5088 explains a novel stockpiling mechanism.

The bacterial type VI secretion system (T6SS) secretes many toxic effectors to gain advantage in interbacterial competition and for eukaryotic host infection. The cognate immunity proteins of these effectors protect bacteria from their own effectors. PldB is a T6SS trans-kingdom effector in Pseudomonas aeruginosa that can infect both prokaryotic and eukaryotic cells. Three proteins, PA5086, PA5087 and PA5088, are employed to suppress the toxicity of PldB-family proteins. The structures of PA5087 and PA5088 have previously been reported, but the identification of further distinctions between these immunity proteins is needed. Here, the crystal structure of PA5086 is reported at 1.90 Šresolution. A structural comparison of the three PldB immunity proteins showed vast divergences in their electrostatic potential surfaces. This interesting phenomenon provides an explanation of the stockpiling mechanism of T6SS immunity proteins.

An enzyme-free immunosensor for sensitive determination of procalcitonin using NiFe PBA nanocubes@TB as the sensing matrix.

In this work, a label-free electrochemical immunosensor was developed for the detection of procalcitonin (PCT), using toluidine blue functionalized NiFe Prussian-blue analog nanocubes (NiFe PBA nanocubes@TB) as a signal amplifier. NiFe PBA nanocubes was synthesized by a simple and efficient self-templating method in this work. NiFe PBA nanocubes with open-framework construction not only provides a larger specific area to load a mass of antibodies but produces an excellent signal without adding extra reaction reagent. Besides, the electrochemical performance of NiFe PBA nanocubes can be enhanced after functionalized with TB. The developed immunosensor exhibited favorable performance for PCT detection with a linear range from 0.001 to 25 ng mL-1 and a detection limit of 3 × 10-4 ng mL-1. Moreover, the immunosensor with acceptable reproducibility, selectivity, and stability may provide a new strategy in the clinical detection of PCT.

Conformational changes of antitoxin HigA from Escherichia coli str. K-12 upon binding of its cognate toxin HigB reveal a new regulation mechanism in toxin-antitoxin systems.

HigA functions as the antitoxin in HigB-HigA toxin-antitoxin system. It neutralizes HigB-mediated toxicity by forming a stable toxin-antitoxin complex. Here the crystal structure of isolated HigA from Escherichia coli str. K-12 has been determined to 2.0 Šresolution. The structural differences between HigA and HigA in HigBA complex imply that HigA undergoes drastic conformational changes upon the binding of HigB. The conformational changes are achieved by rigid motions of N-terminal and C-terminal domains of HigA around its central linker domain, which is different from other known forms of regulation patterns in other organisms. As a transcriptional regulator, HigA bind to its operator DNA through the C-terminal HTH motif, in which key residues were identified in this study.

Crystal structure of the type VI immunity protein Tdi1 (Atu4351) from Agrobacterium tumefaciens.

The type VI secretion system (T6SS) is a novel multiprotein needle-like apparatus that is distributed widely in Gram-negative bacteria. Bacteria harboring T6SSs inject various effectors into both eukaryotic and prokaryotic cells for interspecies competition or virulence-related processes. The toxicities of the effectors can be neutralized by their cognate immunity proteins. Tde1 (Atu4350)-Tdi1 (Atu4351) has recently been characterized as a T6SS effector-immunity pair in the soil bacterium Agrobacterium tumefaciens and the neutralization mechanism remains unknown. Here, the crystal structure of the immunity protein Tdi1 was determined at 2.40 Šresolution by the single-wavelength anomalous dispersion method. Structural analysis suggested that it is composed of a GAD-like domain and an inserted DUF1851 domain, and both domains show low structural similarities to known structures. There is a positive groove mainly located in the GAD-like domain that may be associated with nucleotide binding. The structure provides a basis for further study of the positive groove as a potential active site.

AuCu xO-Embedded Mesoporous CeO2 Nanocomposites as a Signal Probe for Electrochemical Sensitive Detection of Amyloid-Beta Protein.

A sandwich-type electrochemical immunosensor for detecting amyloid-beta protein was fabricated based on Au NP-functionalized reduced graphene oxide (Au@rGO) as an effective sensing platform and AuCu xO-embedded mesoporous CeO2 (AuCu xO@m-CeO2) nanocomposites as the catalytic matrix. The AuCu xO@m-CeO2 composites were obtained by adjusting the amount of m-CeO2 in the reaction to expose enormous active sites. Also, AuCu xO@m-CeO2 was applied as a matrix to immobilize antibodies by forming bridged bonds between m-CeO2 and carboxyl functional groups of antibodies without additional agents. Furthermore, AuCu xO with prominent catalytic activities dramatically improved the performance of the fabricated immunosensor. Also, the morphology, structure, and electronic state of the surface were characterized by SEM, XRD, TEM, and XPS. In addition, the immunosensor demonstrated a wide linear range of 100 fg mL-1 to 10 ng mL-1. This study may provide a way for sensitively detecting various biomarkers.

Facile Synthesis of Cu2O@TiO2-PtCu Nanocomposites as a Signal Amplification Strategy for the Insulin Detection.

Novel ultrasensitive sandwich-type electrochemical immunosensor was proposed for the quantitative detection of insulin, a representative biomarker for diabetes. To this end, molybdenum disulfide nanosheet-loaded gold nanoparticles (MoS2/Au NPs) were used as substrates to modify bare glassy carbon electrodes. MoS2/Au NPs not only present superior biocompatible and large specific surface area to enhance the loading capacity of primary antibody (Ab1) but also present good electrical conductivity to accelerate electron transfer rate. Moreover, the amino-functionalized cuprous oxide decorated with titanium dioxide octahedral composites (Cu2O@TiO2-NH2) were prepared to load dendritic platinum-copper nanoparticles (PtCu NPs) to realize signal amplification strategy. The resultant nanocomposites (cuprous oxide decorated with titanium dioxide octahedral loaded dendritic platinum-copper nanoparticles) demonstrate uniform octahedral morphology and size, which effectively increases the catalytically active sites and specific surface area to load the secondary antibody (Ab2), even increases conductivity. Most importantly, the resultant nanocomposites possess superior electrocatalytic activity for hydrogen peroxide (H2O2) reduction, which present the signal amplification strategy. Under the optimal conditions, the proposed immunosensor exhibited a linear relationship between logarithm of insulin antigen concentration and amperometric response within a broad range from 0.1 pg/mL to 100 ng/mL and a limit detection of 0.024 pg/mL. Meanwhile, the immunosensor was employed to detect insulin in human serum with satisfactory results. Furthermore, it also presents good reproducibility, selectivity, and stability, which exhibits broad application prospects in biometric analysis.

Structural Insights Into the Transcriptional Regulation of HigBA Toxin-Antitoxin System by Antitoxin HigA in Pseudomonas aeruginosa.

HigB-HigA is a bacterial toxin-antitoxin (TA) system in which the antitoxin HigA can mask the endoribonuclease activity of toxin HigB and repress the transcription of the TA operon by binding to its own promoter region. The opportunistic pathogen Pseudomonas aeruginosa HigBA (PaHigBA) is closely associated with the pathogenicity by reducing the production of multiple virulence factors and biofilm formation. However, the molecular mechanism underlying HigBA TA operon transcription by PaHigA remains elusive. Here, we report the crystal structure of PaHigA binding to the promoter region of higBA operon containing two identical palindromic sequences at 3.14 Å resolution. The promoter DNA is bound by two cooperative dimers to essentially encircle the intact palindrome region. The helix-turn-helix (HTH) motifs from the two dimers insert into the major grooves of the DNA at the opposite sides. The DNA adopts a canonical B-DNA conformation and all the hydrogen bonds between protein and DNA are mediated by the DNA phosphate backbone. A higher resolution structure of PaHigA-DNA complex at 2.50 Å further revealed three water molecules bridged the DNA-binding interface and mediated the interactions between the bases of palindromic sequences and PaHigA (Thr40, Asp43, and Arg49). Structure-based mutagenesis confirmed these residues are essential for the specific DNA-binding ability of PaHigA. Our structure-function studies therefore elucidated the cooperative dimer-dimer transcription repression mechanism, and may help to understand the regulation of multiple virulence factors by PaHigA in P. aeruginosa.

Label-free electrochemical immunosensor for insulin detection by high-efficiency synergy strategy of Pd NPs@3D MoSx towards H2O2.

Here, a novel H2O2-based electrochemical immunosensor utilizing Pd nanoparticles functionalized three-dimensional wrinkly amorphous MoSx composites (Pd NPs@3D MoSx) as the platform was developed for the determination of insulin. In this work, Pd NPs@3D MoSx prepared in the presence of CTAB possessed an excellent catalytic activity for the reduction of H2O2. Furthermore, Pd NPs@3D MoSx with favorable biological compatibility can conjugate a great many antibodies to capture insulin. Attributed to the excellent property, electrochemical signals could be greatly amplified, contributing to improving detection sensitivity. Especially, SEM, TEM, and XPS information further confirmed nanomaterial's surface morphology and amorphous structure. Under the optimal conditions, the proposed immunosensor exhibited a sensitively linear relation with logarithmic insulin concentrations from 0.01 to 100 ng/mL with a low detection limit of 3.0 pg/mL (S/N = 3). Characterized by good reproducibility, specificity, and stability, the fabricated immunosensor may blaze a path for insulin detection in a real sample.

Crystal structures of multicopper oxidase CueO G304K mutant: structural basis of the increased laccase activity.

The multicopper oxidase CueO is involved in copper homeostasis and copper (Cu) tolerance in Escherichia coli. The laccase activity of CueO G304K mutant is higher than wild-type CueO. To explain this increase in activity, we solved the crystal structure of G304K mutant at 1.49 Å. Compared with wild-type CueO, the G304K mutant showed dramatic conformational changes in methionine-rich helix and the relative regulatory loop (R-loop). We further solved the structure of Cu-soaked enzyme, and found that the addition of Cu ions induced further conformational changes in the R-loop and methionine-rich helix as a result of the new Cu-binding sites on the enzyme's surface. We propose a mechanism for the enhanced laccase activity of the G304K mutant, where movements of the R-loop combined with the changes of the methionine-rich region uncover the T1 Cu site allowing greater access of the substrate. Two of the G304K double mutants showed the enhanced or decreased laccase activity, providing further evidence for the interaction between the R-loop and the methionine-rich region. The cuprous oxidase activity of these mutants was about 20% that of wild-type CueO. These structural features of the G304K mutant provide clues for designing specific substrate-binding mutants in the biotechnological applications.

The crystal structure of KSHV ORF57 reveals dimeric active sites important for protein stability and function.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a γ-herpesvirus closely associated with Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman disease. Open reading frame 57 (ORF57), a viral early protein of KSHV promotes splicing, stability and translation of viral mRNA and is essential for viral lytic replication. Previous studies demonstrated that dimerization of ORF57 stabilizes the protein, which is critical for its function. However, the detailed structural basis of dimerization was not elucidated. In this study, we report the crystal structures of the C-terminal domain (CTD) of ORF57 (ORF57-CTD) in both dimer at 3.5 Å and monomer at 3.0 Å. Both structures reveal that ORF57-CTD binds a single zinc ion through the consensus zinc-binding motif at the bottom of each monomer. In addition, the N-terminal residues 167-222 of ORF57-CTD protrudes a long "arm" and holds the globular domains of the neighboring monomer, while the C-terminal residues 445-454 are locked into the globular domain in cis and the globular domains interact in trans. In vitro crosslinking and nuclear translocation assays showed that either deletion of the "arm" region or substitution of key residues at the globular interface led to severe dimer dissociation. Introduction of point mutation into the zinc-binding motif also led to sharp degradation of KSHV ORF57 and other herpesvirus homologues. These data indicate that the "arm" region, the residues at the globular interface and the zinc-binding motif are all equally important in ORF57 protein dimerization and stability. Consistently, KSHV recombinant virus with the disrupted zinc-binding motif by point mutation exhibited a significant reduction in the RNA level of ORF57 downstream genes ORF59 and K8.1 and infectious virus production. Taken together, this study illustrates the first structure of KSHV ORF57-CTD and provides new insights into the understanding of ORF57 protein dimerization and stability, which would shed light on the potential design of novel therapeutics against KSHV infection and related diseases.