Molecular Replacement Method Research Articles

Two medicinal molecules Hydralazine and Isoniazid have the potential to control wheat crown rot by combing with a N-acetyltransferase FDB2

Fusarium pseudograminearum is the main pathogen that causes wheat crown rot (WCR), causing serious harm to wheat production. Wheat secretes Benzoxazolinones (Bxs) as fungicidins to prevent F. pseudograminearum infection. Fusarium Detoxification of Bx 2 (FDB2) can degrade Bx to non-fungitoxic N-(2-hydroxyphenyl) malonamic acid. Therefore, FDB2 may be a potential drug target for WCR.In the present study, the structure of FDB2 was determined using the molecular replacement method. The overall FDB2 structure displayed a typical N-acetyltransferase (NAT1) conformation. Unlike other NAT1s, the active site cleft is divided into two parts by a long loop (A135MSPYPDVRKNQA147). Hydralazine, Isoniazid, and 2,4′-dibromoacetanilide were screened out as potential inhibitors of FDB2 by structure alignment. Affinity measurements by MST showed that FDB2 prefers to combine Isoniazid and Hydralazine rather than its natural substrate, 2-aminophenol. Wheat seedling infection assays showed that Isoniazid and Hydralazine suppress F. pseudograminearum invasion in wheat. Our study found that Hydralazine and Isoniazid have the potential to control WCR. This article provides a new idea for the application of medicine, which has serious adverse effects, on plant disease control to reduce research costs and make obsolete drugs shine with vitality.

First crystal structure of the DUF2436 domain of virulence proteins from Porphyromonas gingivalis.

Porphyromonas gingivalis is a major pathogenic oral bacterium that is responsible for periodontal disease. It is linked to chronic periodontitis, gingivitis and aggressive periodontitis. P. gingivalis exerts its pathogenic effects through mechanisms such as immune evasion and tissue destruction, primarily by secreting various factors, including cysteine proteases such as gingipain K (Kgp), gingipain R (RgpA and RgpB) and PrtH (UniProtKB ID P46071). Virulence proteins comprise multiple domains, including the pro-peptide region, catalytic domain, K domain, R domain and DUF2436 domain. While there is a growing database of knowledge on virulence proteins and domains, there was no prior evidence or information regarding the structure and biological function ofthe well conserved DUF2436 domain. In this study, the DUF2436 domain of PrtH from P. gingivalis (PgDUF2436) was determined at 2.21 Å resolution, revealing a noncanonical β-jelly-roll sandwich topology with two antiparallel β-sheets and one short α-helix. Although the structure of PgDUF2436 was determined by the molecular-replacement method using an AlphaFold model structure as a template, there were significant differences in the positions of β1 between the AlphaFold model and the experimentally determined PgDUF2436 structure. The Basic Local Alignment Search Tool sequence-similarity search program showed no sequentially similar proteins in the Protein Data Bank. However, DaliLite search results using structure-based alignment revealed that the PgDUF2436 structure has structural similarity Z-scores of 5.9-5.4 with the C-terminal domain of AlgF, the D4 domain of cytolysin, IglE and the extracellular domain structure of PepT2. This study has elucidated the structure of the DUF2436 domain for the first time and a comparative analysis with similar structures has been performed.

NucleoFind: a deep-learning network for interpreting nucleic acid electron density.

Nucleic acid electron density interpretation after phasing by molecular replacement or other methods remains a difficult problem for computer programs to deal with. Programs tend to rely on time-consuming and computationally exhaustive searches to recognise characteristic features. We present NucleoFind, a deep-learning-based approach to interpreting and segmenting electron density. Using an electron density map from X-ray crystallography obtained after molecular replacement, the positions of the phosphate group, sugar ring and nitrogenous base group can be predicted with high accuracy. On average, 78% of phosphate atoms, 85% of sugar atoms and 83% of base atoms are positioned in predicted density after giving NucleoFind maps produced following successful molecular replacement. NucleoFind can use the wealth of context these predicted maps provide to build more accurate and complete nucleic acid models automatically.

Crystal structures of the 3C proteases from Coxsackievirus B3 and B4.

Enteroviruses cause a wide range of disorders with varying presentations and severities, and some enteroviruses have emerged as serious public health concerns. These include Coxsackievirus B3 (CVB3), an active causative agent of viral myocarditis, and Coxsackievirus B4 (CVB4), which may accelerate the progression of type 1 diabetes. The 3C proteases from CVB3 and CVB4 play important roles in the propagation of these viruses. In this study, the 3C proteases from CVB3 and CVB4 were expressed in Escherichia coli and purified by affinity chromatography and gel-filtration chromatography. The crystals of the CVB3 and CVB4 3C proteases diffracted to 2.10 and 2.01 Å resolution, respectively. The crystal structures were solved by the molecular-replacement method and contained a typical chymotrypsin-like fold and a conserved His40-Glu71-Cys147 catalytic triad. Comparison with the structures of 3C proteases from other enteroviruses revealed high similarity with minor differences, which will guide the design of 3C-targeting inhibitors with broad-spectrum properties.

Structure of a phosphodiesterase from Streptomyces sanglieri with a novel C-terminal domain

A glycerophosphoethanolamine ethanolaminephosphodiesterase (GPE-EP) from Streptomyces sanglieri hydrolyzes glycerophosphoethanolamine to phosphoethanolamine and glycerol. The structure of GPE-EP was determined by the molecular replacement method using a search model generated with AlphaFold2. This structure includes the entire length of the mature protein and it is composed of an N-terminal domain and a novel C-terminal domain connected to a flexible linker. The N-terminal domain is the catalytic domain containing calcium ions at the catalytic site. Coordination bonds were observed between five amino acid residues and glycerol. Although the function of the C-terminal domain is currently unknown, inter-domain interactions between the N- and C-terminal domains may contribute to its relatively high thermostability.

Structure of a novel form of phosphopantetheine adenylyltransferase from Klebsiella pneumoniae at 2.59Å resolution.

Phosphopantetheine adenylyltransferase (EC. 2.7.7.3, PPAT) catalyzes the penultimate step of the multistep reaction in the coenzyme A (CoA) biosynthesis pathway. In this step, an adenylyl group from adenosine triphosphate (ATP) is transferred to 4'-phosphopantetheine (PNS) yielding 3'-dephospho-coenzyme A (dpCoA) and pyrophosphate (PPi). PPAT from strain C3 of Klebsiella pneumoniae (KpPPAT) was cloned, expressed and purified. It was crystallized using 0.1M HEPES buffer and PEG10000 at pH 7.5. The crystals belonged to tetragonal space group P41212 with cell dimensions of a = b = 72.82Å and c = 200.37Å. The structure was determined using the molecular replacement method and refined to values of 0.208 and 0.255 for Rcryst and Rfree factors, respectively. The structure determination showed the presence of three crystallographically independent molecules A, B and C in the asymmetric unit. The molecules A and B are observed in the form of a dimer in the asymmetric unit while molecule C belongs to the second dimer whose partner is related by crystallographic twofold symmetry. The polypeptide chain of KpPPAT folds into a β/α structure. The conformations of the side chains of several residues in the substrate binding site in KpPPAT are significantly different from those reported in other PPATs. As a result, the modes of binding of substrates, phosphopantetheine (PNS) and adenosine triphosphate (ATP) differ considerably. The binding studies using fluorescence spectroscopy indicated a KD value of 3.45 × 10-4M for ATP which is significantly lower than the corresponding values reported for PPAT from other species.

X-ray structure of recombinant house dust mite allergen Der p 3

The X-ray structure of the Der p 3 allergen was solved at 2.25 Å resolution using the molecular replacement method. The most remarkable difference in conformation of the polypeptide chains between the Der p 3 allergen and bovine trypsin is observed in the disordered parts of the polypeptide chain where the degree of homology is lower. The active site of the Der p 3 allergen which involves the residues of catalytic triad Ser197, His 52, and Asp97 (Ser195, His57, and Asp102 in bovine trypsin) responsible for the specific binding of the positively charged substrates Asp191 (Asp189 in bovine trypsin) is in the cleft between the domains.

Preliminary X-ray Diffraction Analysis of Purine Nucleoside Phosphorylase from the Haloalkaliphilic Bacterium Halomonas chromatireducens

Crystals of the enzyme purine nucleoside phosphorylase from the extremophilic bacterium Halomonas Chromatireducens AGD 8-3, suitable for X-ray diffraction, were grown by the vapor-diffusion method. The X-ray diffraction data were collected from these crystals at the Belok beamline of the Kurchatov synchrotron radiation source (National Research Centre “Kurchatov Institute”) at 100 K to 1.80 Å resolution. The X-ray diffraction data were processed in the space groups P1, P2, P21, and P622. The structure was solved by the molecular replacement method taking into account the twinning in the space groups P21 and P1 with one and two hexamers of the enzyme per asymmetric unit, respectively.

Crystal Structure and Sequence Analysis of N5, N10-Methylenetetrahydrofolate Dehydrogenase/Cyclohydrolase Enzyme from Porphyromonas gingivalis

The methylenetetrahydrofolate dehydrogenase–cyclohydrolase (FolD) enzyme has a dual activity of N5,N10-methylenetetrahydrofolate dehydrogenase and cyclohydrolase. This enzyme plays a critical role in the chemical modification of tetrahydrofolate, which is an important coenzyme involved in the synthesis of DNA, RNA, and amino acids. Therefore, bacterial FolD has been studied as a potential drug target for the development of antibiotics. Here, we determined the crystal structure of FolD (PgFolD) from the oral pathogen Porphyromonas gingivalis at 2.05 Å resolution using the molecular replacement method. The crystal structure of PgFolD was successfully refined to a crystallographic R-factor of 21.4% (Rfree = 23.8%). The crystals belong to the space group of P4322 with the unit cell parameters of a = 110.7 Å, b = 110.7 Å, and c = 69.8 Å, containing one subunit in the asymmetric unit. Our analytical size-exclusion chromatography results indicated that PgFolD forms a stable dimer in solution. Additionally, structural and sequence comparison studies with previously known FolDs revealed that PgFolD has a different substrate-binding site residue composition. These findings provide valuable insights for the structure-based development of specific inhibitors against the Porphyromonas gingivalis pathogen.

Crystal structure analysis of pyrrolidone carboxyl peptidase from Thermus thermophilus

Pyrrolidone carboxyl peptidase (PCP) hydrolytically removes the L-pyroglutamic acid from the amino terminal region of pyroglutamyl proteins or peptides. So far, only a limited number of structures of PCP have been solved. Here we report the crystal structure of pyrrolidone carboxyl peptidase from Thermus thermophilus (TtPCP) which has been solved using the molecular replacement method and refined at 1.9 Å resolution. TtPCP follows the α/β/α architecture in which the central β-sheets are surrounded by α-helices on both sides. The inter subunit contact between two monomers consists of two short antiparallel β-strands and part of a long protrusion loop. By comparing the TtPCP with its structural homologs, we identified the putative catalytic triad residues as Glu76, Cys139 and His160. A unique disulfide link found in some homologs of TtPCP, formed between two monomers that provide thermal stability to the protein, is not observed in TtPCP. Hence, being a thermophilic protein, the putative thermal stability of TtPCP could be due to more intra and inter-molecular hydrogen bonds, hydrophobic and ion pair interactions when compared with its mesophilic counterpart. The structural details of TtPCP will be helpful to understand the basis of the intrinsic stability of thermophilic proteins. Also, it could be useful for protein engineering.

Crystal structure and reaction mechanism of a bacterial Mg-dechelatase homolog from the Chloroflexi Anaerolineae.

Chlorophyll degradation plays a myriad of physiological roles in photosynthetic organisms, including acclimation to light environment and nutrient remobilization during senescence. Mg extraction from chlorophyll a is the first and committed step of the chlorophyll degradation pathway. This reaction is catalyzed by the Mg-dechelatase enzyme encoded by Stay-Green (SGR). The reaction mechanism of SGR protein remains elusive since metal ion extraction from organic molecules is not a common enzymatic reaction. Additionally, experimentally derived structural information about SGR or its homologs has not yet been reported. In this study, the crystal structure of the SGR homolog from Anaerolineae bacterium was determined using the molecular replacement method at 1.85 Å resolution. Our previous study showed that three residues-H32, D34, and D62 are essential for the catalytic activity of the enzyme. Biochemical analysis involving mutants of D34 residue further strengthened its importance in the functioning of the dechelatase. Docking simulation also revealed the interaction between the D34 side chain and central Mg ion of chlorophyll a. Structural analysis showed the arrangement of D34/H32/D62 in the form of a catalytic triad that is generally found in hydrolases. The probable reaction mechanism suggests that deprotonated D34 side chain coordinates and destabilizes Mg, resulting in Mg extraction. Besides, H32 possibly acts as a general base catalyst and D62 facilitates H32 to be a better proton acceptor. Taken together, the reaction mechanism of SGR partially mirrors the one observed in hydrolases.

Identification, structure determination and analysis of Mycobacterium smegmatis acyl-carrier protein synthase (AcpS) crystallized serendipitously.

The unintended crystallization of proteins which generally originate from the expression host instead of the target recombinant proteins is periodically reported. Despite the massive technological advances in the field, assigning a structural model to the corresponding diffraction data is not a trivial task. Here, the structure of acyl-carrier protein synthase (AcpS) from Mycobacterium smegmatis (msAcpS), which crystallized inadvertently in an experimental setup to grow crystals of a Mycobacterium tuberculosis protein using M. smegmatis as an expression system, is reported. After numerous unsuccessful attempts to solve the structure of the target protein by the molecular-replacement method no convincing solutions were obtained, indicating that the diffraction data may correspond to a crystal of an artifactual protein, which was finally identified by the Sequence-Independent Molecular replacement Based on Available Databases (SIMBAD) server. The msAcpS structure was solved at 2.27 Å resolution and structural analysis showed an overall conserved fold. msAcpS formed a trimeric structure similar to those of other reported structures of AcpS from various organisms; however, the residues involved in trimer formation are not strictly conserved. An unrelated metal ion (Ni2+), which was possibly incorporated during protein purification, was observed in the proximity of His49 and His116. Structural and sequence differences were observed in the loop connecting the α3 and α4 helices that is responsible for the open and closed conformations of the enzyme. Moreover, the structural analysis of msAcpS augments the current understanding of this enzyme, which plays a crucial role in the functional activation of acyl-carrier proteins in the fatty-acid biosynthesis pathway.

NMR Molecular Replacement Provides New Insights into Binding Modes to Bromodomains of BRD4 and TRIM24.

Structure-based drug discovery (SBDD) largely relies on structural information from X-ray crystallography because traditional NMR structure calculation methods are too time consuming to be aligned with typical drug discovery timelines. The recently developed NMR molecular replacement (NMR2) method dramatically reduces the time needed to generate ligand–protein complex structures using published structures (apo or holo) of the target protein and treating all observed NOEs as ambiguous restraints, bypassing the laborious process of obtaining sequence-specific resonance assignments for the protein target. We apply this method to two therapeutic targets, the bromodomain of TRIM24 and the second bromodomain of BRD4. We show that the NMR2 methodology can guide SBDD by rationalizing the observed SAR. We also demonstrate that new types of restraints and selective methyl labeling have the potential to dramatically reduce “time to structure” and extend the method to targets beyond the reach of traditional NMR structure elucidation.

Elucidation of a nutlin-derivative—HDM2 complex structure at the interaction site by NMR molecular replacement: A straightforward derivation

Protein—ligand complex structures are key in structure-based drug discovery, but their derivation largely relies on X-ray crystallography. While NMR is able to provide atomic resolution complex structures, traditional NMR structure calculation methods are too slow for drug discovery timelines. We recently developed the NMR molecular replacement (NMR2) method that substantially reduces the time needed to derive protein—ligand complex structures, mainly by bypassing the laborious protein sequential resonance assignment step. Here we show how we applied NMR2 to derive the structure of the protein HDM2 in complex with the small molecule caylin-1, an analog of nutlin, based on the HDM2—nutlin complex structure that was already derived by NMR2. This study illustrates how sparse information from a previous NMR2 structure elucidation can be employed to efficiently determine further protein-analog complex structures. We think NMR2 has the potential to become a major tool in structure-based drug discovery, especially when X-ray crystallography is difficult to implement.

A Complexed Crystal Structure of a Single-Stranded DNA-Binding Protein with Quercetin and the Structural Basis of Flavonol Inhibition Specificity.

Single-stranded DNA (ssDNA)-binding protein (SSB) plays a crucial role in DNA replication, repair, and recombination as well as replication fork restarts. SSB is essential for cell survival and, thus, is an attractive target for potential antipathogen chemotherapy. Whether naturally occurring products can inhibit SSB remains unknown. In this study, the effect of the flavonols myricetin, quercetin, kaempferol, and galangin on the inhibition of Pseudomonas aeruginosa SSB (PaSSB) was investigated. Furthermore, SSB was identified as a novel quercetin-binding protein. Through an electrophoretic mobility shift analysis, myricetin could inhibit the ssDNA binding activity of PaSSB with an IC50 of 2.8 ± 0.4 μM. The effect of quercetin, kaempferol, and galangin was insignificant. To elucidate the flavonol inhibition specificity, the crystal structure of PaSSB complexed with the non-inhibitor quercetin was solved using the molecular replacement method at a resolution of 2.3 Å (PDB entry 7VUM) and compared with a structure with the inhibitor myricetin (PDB entry 5YUN). Although myricetin and quercetin bound PaSSB at a similar site, their binding poses were different. Compared with myricetin, the aromatic ring of quercetin shifted by a distance of 4.9 Å and an angle of 31° for hydrogen bonding to the side chain of Asn108 in PaSSB. In addition, myricetin occupied and interacted with the ssDNA binding sites Lys7 and Glu80 in PaSSB whereas quercetin did not. This result might explain why myricetin could, but quercetin could not, strongly inhibit PaSSB. This molecular evidence reveals the flavonol inhibition specificity and also extends the interactomes of the natural anticancer products myricetin and quercetin to include the OB-fold protein SSB.

Sequence Analysis and Preliminary X-ray Crystallographic Analysis of an Acetylesterase (LgEstI) from Lactococcus garvieae

A gene encoding LgEstI was cloned from a bacterial fish pathogen, Lactococcus garvieae. Sequence and bioinformatic analysis revealed that LgEstI is close to the acetyl esterase family and had maximum similarity to a hydrolase (UniProt: Q5UQ83) from Acanthamoeba polyphaga mimivirus (APMV). Here, we present the results of LgEstI overexpression and purification, and its preliminary X-ray crystallographic analysis. The wild-type LgEstI protein was overexpressed in Escherichia coli, and its enzymatic activity was tested using p-nitrophenyl of varying lengths. LgEstI protein exhibited higher esterase activity toward p-nitrophenyl acetate. To better understand the mechanism underlying LgEstI activity and subject it to protein engineering, we determined the high-resolution crystal structure of LgEstI. First, the wild-type LgEstI protein was crystallized in 0.1 M Tris-HCl buffer (pH 7.1), 0.2 M calcium acetate hydrate, and 19% (w/v) PEG 3000, and the native X-ray diffraction dataset was collected up to 2.0 Å resolution. The crystal structure was successfully determined using a molecular replacement method, and structure refinement and model building are underway. The upcoming complete structural information of LgEstI may elucidate the substrate-binding mechanism and provide novel strategies for subjecting LgEstI to protein engineering.

Crystallographic molecular replacement using an in silico-generated search model of SARS-CoV-2 ORF8.

The majority of crystal structures are determined by the method of molecular replacement (MR). The range of application of MR is limited mainly by the need for an accurate search model. In most cases, pre‐existing experimentally determined structures are used as search models. In favorable cases, ab initio predicted structures have yielded search models adequate for MR. The ORF8 protein of SARS‐CoV‐2 represents a challenging case for MR using an ab initio prediction because ORF8 has an all β‐sheet fold and few orthologs. We previously determined experimentally the structure of ORF8 using the single anomalous dispersion (SAD) phasing method, having been unable to find an MR solution to the crystallographic phase problem. Following a report of an accurate prediction of the ORF8 structure, we assessed whether the predicted model would have succeeded as an MR search model. A phase problem solution was found, and the resulting structure was refined, yielding structural parameters equivalent to the original experimental solution.

Affinity and Structural Analysis of the U1A RNA Recognition Motif with Engineered Methionines to Improve Experimental Phasing.

RNA plays a central role in all organisms and can fold into complex structures to orchestrate function. Visualization of such structures often requires crystallization, which can be a bottleneck in the structure-determination process. To promote crystallization, an RNA-recognition motif (RRM) of the U1A spliceosomal protein has been co-opted as a crystallization module. Specifically, the U1-snRNA hairpin II (hpII) single-stranded loop recognized by U1A can be transplanted into an RNA target to promote crystal contacts and to attain phase information via molecular replacement or anomalous diffraction methods using selenomethionine. Herein, we produced the F37M/F77M mutant of U1A to augment the phasing capability of this powerful crystallization module. Selenomethionine-substituted U1A(F37M/F77M) retains high affinity for hpII (KD of 59.7 ± 11.4 nM). The 2.20 Å resolution crystal structure reveals that the mutated sidechains make new S-π interactions in the hydrophobic core and are useful for single-wavelength anomalous diffraction. Crystals were also attained of U1A(F37M/F77M) in complex with a bacterial preQ1-II riboswitch. The F34M/F37M/F77M mutant was introduced similarly into a lab-evolved U1A variant (TBP6.9) that recognizes the internal bulged loop of HIV-1 TAR RNA. We envision that this short RNA sequence can be placed into non-essential duplex regions to promote crystallization and phasing of target RNAs. We show that selenomethionine-substituted TBP6.9(F34M/F37M/F77M) binds a TAR variant wherein the apical loop was replaced with a GNRA tetraloop (KD of 69.8 ± 2.9 nM), laying the groundwork for use of TBP6.9(F34M/F37M/F77M) as a crystallization module. These new tools are available to the research community.

The Glo3 GAP crystal structure supports the molecular niche model for ArfGAPs in COPI coats.

Arf GTPase activating (ArfGAP) proteins are critical regulatory and effector proteins in membrane trafficking pathways. Budding yeast contain two ArfGAP proteins (Gcs1 and Glo3) implicated in COPI coat function at the Golgi, and yeast require Glo3 catalytic function for viability. A new X-ray crystal structure of the Glo3 GAP domain was determined at 2.1Å resolution using molecular replacement methods. The structure reveals a Cys4-family zinc finger motif with an invariant residue (R59) positioned to act as an "arginine finger" during catalysis. Comparisons among eukaryotic GAP domains show a key difference between ArfGAP1 and ArfGAP2/3 family members in the final helix located within the domain. Conservation at both the sequence and structural levels suggest the Glo3 GAP domain interacts with yeast Arf1 switch I and II regions to promote catalysis. Together, the structural data presented here provide additional evidence for placing Glo3 near Arf1 triads within membrane-assembled COPI coats and further support the molecular niche model for COPI coat regulation by ArfGAPs.

Crystal structure and mutational analysis of the human TRIM7 B30.2 domain provide insights into the molecular basis of its binding to glycogenin-1

Tripartite motif (TRIM)7 is an E3 ubiquitin ligase that was first identified through its interaction with glycogenin-1 (GN1), the autoglucosyltransferase that initiates glycogen biosynthesis. A growing body of evidence indicates that TRIM7 plays an important role in cancer development, viral pathogenesis, and atherosclerosis and, thus, represents a potential therapeutic target. TRIM family proteins share a multidomain architecture with a conserved N-terminal TRIM and a variable C-terminal domain. Human TRIM7 contains the canonical TRIM motif and a B30.2 domain at the C terminus. To contribute to the understanding of the mechanism of action of TRIM7, we solved the X-ray crystal structure of its B30.2 domain (TRIM7B30.2) in two crystal forms at resolutions of 1.6 Å and 1.8 Å. TRIM7B30.2 exhibits the typical B30.2 domain fold, consisting of two antiparallel β-sheets of seven and six strands, arranged as a distorted β-sandwich. Furthermore, two long loops partially cover the concave face of the β-sandwich defined by the β-sheet of six strands, thus forming a positively charged cavity. We used sequence conservation and mutational analyses to provide evidence of a putative binding interface for GN1. These studies showed that Leu423, Ser499, and Cys501 of TRIM7B30.2 and the C-terminal 33 amino acids of GN1 are critical for this binding interaction. Molecular dynamics simulations also revealed that hydrogen bond and hydrophobic interactions play a major role in the stability of a modeled TRIM7B30.2-GN1 C-terminal peptide complex. These data provide useful information that could be used to target this interaction for the development of potential therapeutic agents.