Current Research in Structural Biology

Biosynthesis of phospholipids is fundamental for membrane biogenesis in all living organisms. As a member of the Glycerol-3-phosphate (G3P) Acyltransferase (GPAT) family, PlsB is a crucial enzyme catalyzing the first step of phospholipid synthesis by converting G3P and fatty acyl-coenzyme A (CoA)/acyl-carrier protein (ACP) into lysophosphatidic acid and free CoA (CoASH)/ACP. In bacterial cells, PlsB participates in the formation of persister cells related to multidrug tolerance, and is hence considered as a potential target for anti-persister therapy. By using the single-particle cryo-electron microscopy (cryo-EM) method, we have solved the structure of full-length PlsB from Themomonas haemolytica (ThPlsB) at 2.79 [A] resolution. The ThPlsB protein forms a homodimer with C2 symmetry and each monomer contains three distinct domains, namely the amino-terminal domain (NTD), the middle catalytic domain (MCD) and the carboxy-terminal domain (CTD). For the first time, we have unraveled the binding sites of a fatty acyl-CoA and a 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) molecule in the MCD of PlsB. The interactions between ThPlsB and the membrane involve two surface-exposed amphipathic regions located in the NTD and MCD respectively. The results of structural and biochemical analyses suggest a membrane surface association-catalysis coupling model for the PlsB-mediated biosynthesis of lysophosphatidic acid occurring at the membrane-cytosol interface.

Class II Fructose-1,6-bisphosphatases (FBPaseII) (EC: 3.1.3.11) are highly conserved essential enzymes in the gluconeogenic pathway of microorganisms. Previous crystallographic studies of FBPasesII provided insights into various inactivated states of enzymes from different species. Presented here is the first crystal structure of FBPaseII in an active state, solved for the enzyme from Francisella tularensis (FtFBPaseII), containing native metal cofactor Mn2+ and complexed with catalytic product fructose-6-phosphate (F6P). Another crystal structure of the same enzyme complex is presented in inactivated state due to the structural changes introduced by crystal packing. Analysis of the interatomic distances among the substrate, product and divalent metal cations in the enzyme catalytic centers led to a revision of the catalytic mechanism suggested previously for class II FBPases. Instead of a metal cofactor for the stabilization of the transition state of the leaving phosphate group, we propose that the positive dipole of the neighboring -helix backbone (G88-T89-T90-I91-T92-S93-K94) is responsible for retaining the cleaved phosphate. The revised catalytic mechanism involves a nucleophilic attack by a reactive water coordinated by both the T89 hydroxyl and a second water molecule coordinated directly by Mn2+. Additionally, a crystal structure of Mycobacterium tuberculosis FBPaseII (MtFBPaseII), containing the substrate fructose-1,6-bisphosphate (F1,6BP) bound, is presented in support of a novel catalytic mechanism for this class of enzymes.

MHETases are enzymes implicated in polyethylene terephthalate (PET) biodegradation. The present study elucidates the structural determinants that result in increased mono(2-hydroxyethyl) terephthalate (MHET) degradation by a feruloyl esterase, which has been engineered to resemble MHETase active site. The crystal structures of the variant in apo and benzoic acid bound state reveal the changes induced by the introduced mutation, specifically the formation of a hydrogen bond and a trans to cis isomerization of a peptide bond in the vicinity of the catalytic site. Molecular dynamics simulations demonstrate the stabilization of the loop harboring the engineered residue, as well as an expansion of the substrate binding cleft, which would facilitate accommodation of a broader variety of substrates, indicative of a promiscuous biocatalyst. EnzymeEnzyme Commission Number: EC 3.1.1.73; Uniprot accession code: A0A1D3S5H0_FUSOX

1Polymyxins are last-resort antimicrobial peptides administered clinically against multi-drug resistant bacteria, including Gram-negative ESKAPE pathogens. However, an increasing number of pathogens employ a defense strategy involving a relay of enzymes encoded by the pmrE(ugd) loci and the arnBCDTEF operon. As a result, an Ara-4N headgroup is added to the lipid-A component of outer membrane (OM) lipopolysaccharides (LPS) rendering polymyxins ineffective. Here, we report the cryo-EM structures of glycosyltransferase ArnC from Salmonella typhimurium resolved in both apo and UDP-bound forms at resolutions 2.75 [A] and 3.8 [A], respectively. The structure of the ArnC protomer comprises of three distinct regions: an N-terminal glycosyltransferase domain, transmembrane region, and the interface helices (IHs). ArnC forms a stable tetramer with C2 symmetry through interactions in the C-terminal region, which is expected to protrude into the cytosol, where the {beta}8 strand inserts into the adjacent protomer. ArnC protomers have two distinct types of interfaces involving multiple hydrogen bonds and salt bridges. The binding of UDP induces conformational changes that stabilizes structurally labile A-loop, spanning residues 201 to 213, and part of the putative catalytic pocket formed by IH1 and IH2. The comparative analysis of ArnC structures with homologs GtrB and DPMS suggests the key residues involved in ArnC catalytic activity.

Succinimide (SNN), an intermediate formed during asparaginyl deamidation or aspartyl dehydration in proteins, is generally hydrolysis-prone, leading to isomerization to L/D /{beta}-aspartyl residue, with the latter being considered deleterious to protein structure and function. An unusually stable SNN-mediated conformational rigidity through restriction of the backbone dihedral angle, {psi}, enhances the thermostability of glutamine amidotransferase (GATase) from Methanocaldococcus jannaschii (Mj). Although several structural features involved in maintaining a stable SNN and imparting SNN-mediated thermostability have been identified in MjGATase, the residues in the protein that catalyse the rapid and complete conversion of Asn109 to SNN remain unknown. Here, we investigated several site-directed mutants of MjGATase for their ability to retain Asn109 side chain in the unmodified form. Mass spectrometric analysis of 10 single mutants enabled the identification of residues that impacted the proportion of SNN and Asn population in the protein sample. This led to the generation of two double mutants that retained intact Asn109 side chain as observed in the mass spectra and crystal structures. These mutants with intact Asn residue at position 109, displayed lower thermal stability than the protein with the SNN modification. Further understanding of the deprotonation mechanism was addressed using QM/MM MD metadynamics simulations. HighlightsO_LIStable succinimide (SNN) arising from deamidation of Asn109 residue imparts hyperthermostability to MjGATase. C_LIO_LIExamination of the structure of MjGATase suggests neighbouring residues playing possible roles in deamidation and cyclization. C_LIO_LIExamination by LC-MS of single site-directed mutants of residues contacting SNN revealed varied levels of intact Asn109 enabling generation of double mutants with complete absence of deamidation. C_LIO_LIPresence of intact Asn109 confirmed by X-ray crystallography highlights the role of Y158, D110, and K151 in mediating SNN formation. C_LIO_LIQM/MM MD metadynamics simulations support experimental findings. C_LI

The thaumarchaeal 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle represents one of the most efficient mechanisms for CO2 fixation discovered to date. Within this cycle, the enzyme encoded by Nmar_1308 from Nitrosopumilus maritimus SCM1 plays a crucial role due to its dual functionality as both a crotonyl-CoA hydratase (CCAH) and a 3-hydroxypropionyl-CoA dehydratase (3HPD). Although the importance of a bifunctional enzyme for lowering the cost of biosynthesis, the details of structural dynamics are still missing. Here, in addition to our cryogenic temperature structures, we determined the first ambient temperature structures of the Nmar_1308 protein by Serial Femtosecond X-ray Crystallography (SFX). The determined structures capture previously unobserved conformational dynamics of the Nmar_1308 protein, providing invaluable information for future synthetic biology applications.

While vaccines have by large been found to effective against the evolving SARS-CoV-2 variants, the profound and rapid effectivity of monoclonal antibodies (mAbs) in significantly reducing hospitalization to severe disease outcomes have also been demonstrated. In the present study, by high resolution cryo-electron microscopy (cryo-EM), we examined the structural insights of two trimeric spike (S) protein bound mAbs isolated from an Indian convalescent individual infected with ancestral SARS-CoV-2 which we recently reported to potently neutralize SARS-CoV-2 from its ancestral form through highly virulent Delta form however different in their ability to neutralize Omicron variants. Our findings showed binding and conformational heterogeneities of both the mAbs (THSC20.HVTR04 and THSC20.HVTR26) bound to S trimer in its apo and hACE-2 bound forms. Additionally, cryo-EM resolved structure assisted modeling highlighted key residues associated with the ability of these two mAbs to neutralize Omicron variants. Our findings highlighted key interacting features modulating antigen-antibody interacting that can further aid in structure guided antibody engineering to enhance their breadth and potency. HighlightsO_LITwo potent human mAbs obtained from a single donor differ binding to Omicron spikes C_LIO_LIPattern of binding and conformation of these mAbs bound to full length spike differs C_LIO_LIAntibody binding alters the conformational states of S trimer in its apo and hACE-2 bound forms. C_LIO_LICryo-EM structure guided modeling highlighted correlates of interacting residues associated with resistance and sensitivity of BA.1, BA.2, BA.4/BA.5 resistance and sensitivity against these mAbs. C_LI

Cyclic-di-nucleotide based secondary messengers regulate various physiological processes including the stress responses in bacteria. In the past decade, cyclic diadenosine monophosphate (c-di-AMP) has emerged as a crucial second messenger, implicated in fatty acid metabolism, antibiotic resistance, biofilm formation, virulence, DNA repair, ion homeostasis, sporulation etc. The level of c-di-AMP is maintained in the cell by the action of two opposing enzymes, namely diadenylate cyclase (DAC) and phosphodiesterase (PDE). In mycobacteria, this molecule is essential for its regulatory role in bacterial physiology and host-pathogen interactions. However, such modulation of c-di-AMP remains to be explored in Mycobacterium smegmatis. Here, we systematically characterised the c-di-AMP synthase (MsDisA) and a hydrolase (MsPDE) from M. smegmatis at different pH and osmolytic conditions in vitro. Our biochemical assays show that the MsDisA activity is enhanced during the alkaline stress and c-di-AMP is readily produced without any intermediates. At pH 9.4, the MsDisA promoter activity in vivo increases significantly, strengthening this observation. However, under physiological conditions, the activity of MsDisA was moderate with the formation of intermediates. To get further insights into the structural characteristics, we determined the cryo-EM structure of the MsDisA, revealing some interesting features. Biochemical analysis of individual domains shows that the N-terminal minimal region alone can form a functional octamer. Altogether, our results reveal the biochemical and structural regulation of mycobacterial c-di-AMP in response to various environmental stress.

MgtE channels play crucial roles in Mg{superscript 2} homeostasis and are implicated in bacterial survival under antibiotic exposure. Previous structural and biophysical studies have predominantly focused on the Thermus thermophilus MgtE, leaving the structural and mechanistic diversity of MgtE family proteins largely unexplored. In this study, using a genome mining approach, we identified diverse MgtE homologs, including a novel subclass termed the "mini-N type," which lacks the canonical cytoplasmic N and CBS domains but possesses a unique small N-like domain. Despite extensive expression screening, mini-N type homologs could not be stably purified. To address this issue, we designed a series of de novo proteins and determined their crystal structures. A selected de novo protein was fused to a mini-N type MgtE, enabling successful purification and preliminary cryo-EM imaging. Our findings demonstrate that de novo designed protein fusions can serve as powerful tools for stabilizing and purifying otherwise unstable membrane proteins, opening new avenues for the structural and functional studies of otherwise inaccessible membrane proteins.

Mycobacterium tuberculosis isocitrate lyase 2 (ICL2) is an allosterically regulated enzyme that enables the bacterium to survive on non-glycolytic substrates during infection. Previous studies showed that ICL2 is allosterically regulated by acetyl-CoA and its analogues but the molecular mechanism underpinning this regulation is unknown. Here, we use protein NMR, crystallography, molecular dynamics, and mutagenesis studies to show that two unique structural features of ICL2, its C-terminal domain and a unique helical substructure on its N-terminal catalytic domain, play important roles in the enzymes allostery. In particular, we found that the binding of acetyl-CoA promotes the dimerisation of the C-terminal domain and disrupts its interactions with the unique helical substructure on the N-terminal domain. This leads to conformational changes in the ICL2 enzyme that induces activation. Taken together, our findings reveal, for the first time, how the binding of acetyl-CoA, which is not an ICL2 substrate, induces ICL2 activation. By extension, the work also identifies a novel allosteric mechanism controlling M. tuberculosis metabolism that is amenable to therapeutic manipulation. Significance StatementMycobacterium tuberculosis isocitrate lyase 2 (ICL2) was previously shown to be activated by acetyl-CoA and propionyl-CoA - two central metabolites generated by the metabolism of sugars and fatty acids. However, it is not known how the binding of these metabolites leads to the activation of ICL2. Together with its isoform ICL1, ICL2 has been shown to be essential for the survival and pathogenesis of the bacterium. Understanding how this regulation occurs can help design novel treatments to target this protein and eradicate these bacteria, which cause the most deaths worldwide due to a single bacterial agent. This system also presents a fascinating model to examine allostery in proteins, with the techniques illustrated in this paper being applicable to other allosteric proteins.

{gamma}-Glutamyl peptidase 1 (GGP1) plays a dual role in primary and secondary sulfur metabolism in Arabidopsis thaliana. During glutathione (GSH) turnover, GGP1 hydrolyzes the isopeptide bond of GSH to degrade the tripeptide into Glu and Cys-Gly. During glucosinolate and camalexin biosynthesis, GGP1 processes GSH conjugates, which have a large substituent at the thiol side chain, by hydrolyzing the same isopeptide bond of {gamma}-Glu. In the present study, we determined the crystal structures of the following GGP1 forms: ligand-free, Glu complex, covalent {gamma}-Glu intermediate, and disulfide-linked S-S inactive forms. The intermediate structure, in which {gamma}-Glu is covalently linked to the nucleophile C100, was trapped by mutating the catalytic His to Asn (H192N). In the Glu complex and {gamma}-Glu intermediate structures, Glu bound to the S1 subsite is extensively recognized by several hydrogen bonds. The substrate recognition of the Cys-Gly moiety at the S1 and S2 subsites was revealed by modeling GSH in the active site. Mutational analysis indicated that R206 plays an important role in substrate binding by forming a salt bridge with Gly at the S2 subsite. An open pocket is present beyond the thiol side chain of Cys in the S1 subsite, which contributed to the dual activity of GGP1 toward GSH and GSH conjugates. The S-S inactive structure was obtained by soaking GGP1 crystals in Cys-Gly, and the catalytic cysteine (C100) partially formed a disulfide bond with a neighboring C154 residue. The partial inactivation of GGP1 in the presence of a pro-oxidant (Cys-Gly) has revealed its possible role in oxidative stress regulation in Arabidopsis.

Glutamate dehydrogenase (GDH) is a key enzyme in all living organisms and some of the GDHs exhibit substrate-dependent homotropic cooperativity. However, the mode of allosteric communication during the homotropic effect in GDHs remains poorly understood. In this study, we examined two homologous GDHs, Aspergillus niger GDH (AnGDH) and Aspergillus terreus GDH (AtGDH), with differing substrate utilization kinetics to uncover the factors driving their distinct behavior. The crystal structures and first-ever cryo-EM structures of apo-AtGDH captured arrays of conformational ensembles. Comparative structural analysis has revealed a wider mouth opening in allosteric AnGDH ([~] 21 [A]) compared to non-allosteric AtGDH ([~]17 [A]) in their apo states. A network of interaction related to the amino acid substitutions in Domain II is responsible for differential structural dynamics in these GDHs. Remarkably, we identified one remotely located substitution in Domain II, i.e., R246 to S, a part of the network, which reversed the kinetic properties of AtGDH into an allosteric one and controls the mouth opening. Our data also indicate that dynamic discrepancy influences the substrate binding affinity and catalytic activity in AnGDH and AtGDH. We have successfully demonstrated for the first time, that remotely located residues and the conformational dynamics regulate the kinetic properties in homologous GDHs.

Nonribosomal peptide synthetases (NRPSs) are large multidomain enzymes for the synthesis of a variety of bioactive peptides in a modular and pipelined fashion. Here, we investigated how the condensation (C) domain and the adenylation (A) domain cooperate with each other for the efficient catalytic activity in microcystin NRPS modules. We solved two crystal structures of the microcystin NRPS modules, representing two newly identified conformations in the NRPS catalytic cycle. Our data reveals that the dynamic interaction between the C and the A domains in these modules are mediated by the conserved "RXGR" motif, and this interaction is important for the adenylation activity. Furthermore, the "RXGR" motif-mediated dynamic interaction and its functional regulation is prevalent in different NRPSs modules possessing both the A and the C domains. This study provides new insight into the catalytic mechanism of NRPSs and should inspire novel ideas in NRPS enzyme engineering in synthetic biology.

Biosynthetic gene clusters (BGCs) are contiguous genomic regions that encode diverse, non-homologous proteins required for the production of specific natural products. Their genetic diversity underlies the structural complexity of these compounds, and their biosynthetic pathways still remain to be clarified in many cases. The biosynthetic mechanisms rely not only on substrate specificity between proteins and ligands, but also on protein-protein interactions that mediate transport of intermediates, regulation of activity, and structural stabilization. However, sequence-based functional predictions have had limited success for many uncharacterized proteins within BGCs. To address this challenge, we built a high-throughput complex prediction pipeline by replacing AlphaFold3s multiple sequence alignment generation with a faster MMSeqs2. We systematically screened 487,828 protein pairs derived from 2,437 BGCs registered in the Minimum Information about a Biosynthetic Gene cluster (MIBiG) database and predicted 15,438 heteromeric interactions with an ipTM [≥] 0.6. Among them, 1,390 protein pairs exhibited structural homology with an RMSD [≤] 2.0 [A]. These predictions highlight intriguing molecular mechanisms involving proteins previously annotated as uncharacterized or potentially dysfunctional. Furthermore, we provide these results in a reusable format and as a protein interaction network map to facilitate future experimental validation by researchers.

The majority of phages, viruses that infect prokaryotes, deliver their genomic material into host cells via a tail structure. Previous research has established that the central tube of these phages, composed of tail tube protein (TTP), is structurally conserved, typically forming either hexameric or trimeric rings. In this study, we revealed a novel pentameric assembly of TTP and present two cryo-EM structures at resolutions of 3.5 [A] and 3.7 [A], respectively. Our detailed structural analysis demonstrates that the inner surface of the pentameric tube is highly negatively charged. Critical residues located on the loop between {beta}3 and {beta}4 play pivotal roles in the formation of the pentameric rings, and mismatches of the interactions between the stacked layers can induce the curvature of the tube. The cryo-EM structure of the TTP polymer at the tubes end uncovered that the {beta}-strands that span amino acids 27-65 move towards the central tunnel, potentially blocking the tunnel through which the phage genome is released. Our study provides new structural insights into a novel assembly of TTP, which will extend the understanding of phage assembly.

The E. coli cytochrome bo3 ubiquinol oxidase is a four-subunit heme-copper oxidase that serves as a proton pump in the E. coli aerobic respiratory chain. Despite many mechanistic studies on this protein, it is unclear whether this ubiquinol oxidase functions as a monomer, or as a dimer in a manner similar to its eukaryotic counterparts - the mitochondrial electron transport complexes. In this study, we determined the monomeric and dimeric structures of the E. coli cytochrome bo3 ubiquinol oxidase reconstituted in amphipol by cryogenic electron microscopy single particle reconstruction (cryo-EM SPR) to a resolution of 3.15 [A] and 3.46 [A], respectively. We have discovered that the protein can form a dimer in C2 symmetry, with the dimerization interface maintained by interactions between the subunit II of one monomer and the subunit IV of the other monomer. Moreover, the dimerization does not induce significant structural changes in each monomer, except the movement of a loop in subunit IV (residues 67-74).

Pathogenic Escherichia coli (E. coli) causes serious illnesses in humans aided by several multiple antibiotic resistance regulator (MarR) transcription factors. Among these proteins, HosA provides support to these organisms by executing crucial roles in functions ranging from flagella motility to organic compound metabolism. The crystal structure of HosA from enteropathogenic E. coli is elucidated in this study along with conformational changes orchestrated by different amino acids due to para-hydroxy benzoic acid (PHB) binding in the hinge region. Structural analysis and extensive molecular dynamics simulation reveal role of a dynamic water molecule as a bridging entity in PHB bound structure which is not shown for any MarR structure yet. Also, it is shown that the HosA gene is transferred horizontally from Shigella to pathogenic E. coli, having 97.6% sequence similarity with an uncharacterized transcription factor from Shigella dysenteriae Sd197. This study may be promising to address several unanswered questions for the functioning of MarR transcription factors from infectious E. coli and design inhibitors to combat these pathogens.

Leptospires are zoonotic pathogens that cause significant socio-economic burden in developing countries, world-wide. The pathogenic species Leptospira interrogans (Li) is an important and interesting target for investigating the enzymes essential to its metabolic needs and adaptations. We cloned and expressed triosephosphate isomerase (LiTIM), a central metabolic flux regulator of Li, in AA200, E. coli TIM null strain. LiTIM was obtained as an active dimer (D-GAP[->]DHAP kcat = 1740 s-1 and Km (D-GAP) = 0.21 mM, at 25 {degrees}C) with mid-transition concentrations, Cm, 0.8 mM and 2.6 mM, respectively, for guanidine hydrochloride and urea induced equilibrium unfolding. We report the high resolution X-ray structures of LiTIM in apo and substrate (DHAP) bound forms. Our analysis highlights key features of TIM that regulate the mode of substrate binding and transition state stabilization and thus play a decisive role in attainment of high proficiency for the isomerisation reaction while avoiding the elimination reaction. Unexpected differences in the effect of temperature on stability and activity were observed for the three mesophilic pathogenic TIMs viz. from Li, Plasmodium falciparum (Pf) and Trypanosoma brucei (Tb). LiTIM and TbTIM (Tm = 46.5 {degrees}C) were more susceptible to unfolding and precipitation compared to PfTIM (Tm = 67 {degrees}C). In contrast, the initial (or zero point) activity of PfTIM rises till 50 {degrees}C and saturates unlike LiTIM and TbTIM which show a rise till 55 {degrees}C and 60 {degrees}C, respectively. These observations could be rationalized by sequence comparison and examination of the structures of the three TIMs.

Mycobacterium tuberculosis contains thirteen Mycobacterial membrane protein Large (MmpL) transporters, which belong to the family of secondary active RND transporters. MmpL4 and MmpL5, together with their operon partners MmpS4 and MmpS5, export the mycobacterial siderophore mycobactin and the last resort TB drug bedaquiline. Recently, we determined a structure of the MmpL4 monomer in complex with desferrated mycobactin, which lacked a functionally essential coiled-coil domain predicted to extend far into the periplasm. Here, we present a cryo-EM structure of the hexameric (MmpS4)3-(MmpL4)3 complex, which was enabled by rational disulfide cross-links based on AlphaFold predictions. We observed density for the coiled-coil domain, which protrudes into the periplasmic space at an angle of around 60{degrees} relative to the symmetry axis of the MmpL4 trimer. In the context of the hexameric complex, MmpL4s conformation differs strikingly from the one observed for monomeric MmpL4, which includes formation of a large cavity in the periplasmic domain and rearrangements of conserved proton coupling residues at the transmembrane domain. Our work provides an experimental workflow to obtain single particle cryo-EM structures of labile multiprotein complexes by AlphaFold-informed stabilization of predicted protein interfaces.

Ubc13 is a critical ubiquitin-conjugating enzyme involved in the nuclear factor-{kappa}B (NF-{kappa}B) signalling pathway. The Shigella flexneri effector OspI targets the host Ubc13 and modifies this enzyme by deamidation of Gln100 into Glu100. This modification inhibits the tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6)-catalyzed ubiquitination and diacylglycerol-CBM (CARD-Bcl10- Malt1)-TRAF6-NF-{kappa}B signal activation. We have previously reported the wild-type OspI crystal structure, but the catalytic triad does not form the canonical active site. Here, the crystal structure of OspI with a C62S mutation was determined at a resolution of 2.2 [A]. This C62S mutant structure provided the active site conformation with the catalytic site of OspI.