We have determined the X-ray crystallographic protein structure of endo-1,4-β-xylanase (EX) A from Anaerobacterium chartisolvens (AchXyn30A), a homologue of the recent biochemically characterized glycoside hydrolase family 30, subfamily 12 (GH30_12) EX from Acetivibrio clariflavus (AcXyn30B). The N-terminal GH30 catalytic domains (CDs) of these two enzymes share approximately 63% amino-acid sequence identity and the full-length proteins each consist of the GH30_12 CD, a family 6 carbohydrate-binding module and a C-terminal dockerin domain. In this report, we offer additional support for the recent subfamily classification of these EXs and provide detailed X-ray crystallographic protein structure analysis of AchXyn30A, the first protein structure from this newly defined GH30 subfamily. We also provide comparative structural analysis using a generated AcXyn30B homology model as well as other GH30 subfamily enzymes. Additionally, we examine potential xylan-chain interactions informed by the protein structure. These characterized EXs further illustrate the diversity of xylan-degrading enzymes which have evolved within glycoside hydrolase family 30.

Serine-aspartate repeat-containing protein D (SdrD) is a Staphylococcus aureus cell wall-anchored, calcium-binding adhesin member of the MSCRAMM Sdr subfamily that may contribute to bacterial adhesion and virulence. S. aureus is the most common cause of periprosthetic joint infection (PJI). Population-level distribution and sequence diversity of SdrD among clinical PJI isolates have not been systematically characterized, and the SdrD binding mechanism is still not well understood. To address these gaps, sdrD alleles were queried across 156 newly sequenced PJI isolates and compared to publicly available S. aureus genomes, and nucleotide- and protein-level phylogenies of the sdrCDE locus constructed. The SdrD crystal structure from S. aureus JH1 was determined, with solution small-angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, and assessment of conformational changes with calcium depletion. Three dominant sdrD subtypes were defined, associating with USA300, JH1, and TCH60; the JH1 sdrD subtype was predominant among PJI isolates. Structural studies showed that the conformation of individual domains and interdomain organization of the multidomain SdrD have limited flexibility in solution, and that the calcium-binding B domain retains its core fold under conditions of calcium depletion. Together, the findings presented support functional diversification among Sdr family members in mediating host attachment and inform a re-evaluation of the ligand-binding mechanism previously proposed for SdrD.

Proteins that bind to a target protein of interest, termed "binders", are essential components of biological research reagents and therapeutics. Target proteins present multiple binding surfaces with varying interaction potential. "Hot spots", or high-potential surfaces, are experimentally identified as the most probable binding sites in de novo discovery campaigns. However, hot spots and their default binding modes do not always confer the desired specificity. Related proteins or isoforms often share similar hot spots, resulting in promiscuous binding. Interaction with a hot spot may also fail to elicit the intended biological outcome. Consequently, methods that direct de novo binder discovery toward targets with defined specificity are critically needed. We recently developed phage-assisted noncontinuous selection of binders (PANCS-Binders), a selection platform with unparalleled speed and sequence-function fidelity that enables routine de novo binder discovery within days. However, because PANCS-Binders selections enrich variants based primarily on affinity, secondary screening is unlikely to identify binders to lesser hot spots because of the high likelihood of convergence. These alternative binding surfaces with weaker inherent interactions may possess desirable specificity profiles. Here, we develop PANCS-spec-Binders, which incorporates simultaneous selection and counterselection to control the specificity of enriched binders. We demonstrate PANCS-spec-Binders in two proof-of-concept applications: 1) discovery of isoform-selective binders that bind HRAS with >100-fold higher affinity than the highly related KRAS isoform, and 2) discovery of epitope-specific binders that either target or avoid the LIR interaction region of LC3B. PANCS-spec-Binders enables rapid identification of binders with defined specificity within days.

Many Gram-negative bacterial species use contact-dependent growth inhibition (CDI) systems to deliver toxic proteins into neighboring competitors. CDI + strains deploy CdiA effector proteins, which translocate their C-terminal toxin (CT) domains into target bacteria through a receptor-mediated delivery pathway. To protect against auto-intoxication, CDI + bacteria also produce CdiI immunity proteins that neutralize CT toxin activity. Here, we present the crystal structure of the CT·CdiI O32:H37 complex from Escherichia coli O32:H37. CT O32:H37 adopts the same fold as the tRNase domain of colicin D, and the nucleases share similar catalytic centers. However, unlike colicin D, which cleaves the anticodon loops of tRNA Arg isoacceptors, CT O32:H37 exhibits nonspecific RNase activity. Notably, we find that endogenous elongation factor Tu (EF-Tu) co-purifies with the over-produced CT·CdiI O32:H37 complex. Although EF-Tu does not bind stably to CT O32:H37 in the absence of CdiI O32:H37 , the translation factor is required for toxic RNase activity in vitro. AlphaFold 3 modeling and site-directed mutagenesis indicate that CT O32:H37 interacts with the N-terminal GTPase domain of EF-Tu. EF-Tu appears to stabilize residue Trp52 within the hydrophobic core of the toxin, which in turn supports the RNase active site through an unusual hydrogen-bonding interaction with the catalytic His67 residue. Thus, EF-Tu is hijacked as an essential co-factor to organize the toxin's catalytic center.

Antibiotic resistance remains a leading cause of severe infections worldwide. Small changes in protein sequence can impact antibiotic efficacy. Here, we report deposition of 58 X-ray crystal structures of bacterial proteins that are known targets for antibiotics, which expands knowledge of structural variation to support future antibiotic discovery or modifications.

Pathogenic members of the genus Corynebacterium cause a wide range of serious infections in humans including diphtheria. Adhesion to host cells is a crucial step during infection. In Corynebacterium diphtheriae, adhesion is mediated by filamentous structures called pili or fimbriae that are covalently attached to the bacterial cell wall. Pilus assembly proceeds by transpeptidation reactions catalyzed by sortases, followed by covalent anchoring of the filament in the peptidoglycan layer.There are 6 sortases in C. diphtheriae, SrtA, SrtB, SrtC, SrtD, SrtE and SrtF. Five of these proteins are devoted to the assembly of three distinct types of pilus fibres: SrtA for the SpaA-type pilus, SrtB/SrtC for the SpaD-type pilus, and SrtD/SrtE for the SpaH-type pilus. SrtF, the so-called housekeeping sortase, catalyses the cell wall anchoring of pilin monomers as well as pili, but it does not polymerize pilins.We have previously determined structures of the SrtA and SrtF sortases and deposited to Protein Data Bank (PDB). There are no structures of remaining four C. diphtheriae sortases available. Also, biological data for these proteins are sparse. The presence of two sortase pairs involved in the SpaD-and SpaH-type pilus assembly is intriguing. The SrtB/SrtC sortases are able to polymerize SpaDF pili consisting of SpaD and SpaF proteins. However, only SrtB is capable of incorporation of SpaE accessory protein into the SpaD-type pili. There is even less data for SrtD/SrtE pair (40% amino acid identity between these proteins). It was shown that both proteins participate in formation of SpaHIG pili (SpaH-type pili including two accessory proteins SpaI and SpaG). Deletion of either one of sortases results in significantly slower polymerization of SpaH-type pili. No clear difference between SrtD and SrtE was reported.We crystallized and solved the high-resolution structures of four remaining C. diphtheriae sortases: SrtB, SrtC, SrtD and SrtE. The structures, with exception of SrtD, closely resemble Alphafold prediction models. We did not find significant differences between SrtB and SrtC sortase main bodies and active centers. The major difference between SrtB and SrtC is the presence SrtB extended proline-rich 18-residue loop replacing short 3-residue fragment in the SrtC. We think this loop is indirectly involved in the SrtB activity. Also, for SrtD and SrtE sortase pair, we did not find significant differences in core structures and active centers. Nevertheless, we found a unique conformation of the N-terminal helix not predicted by Alphafold models. The helix of the experimental structure is rotated by 180 degrees relatively to the protein main body. We do not exclude possibility that it is a result of crystal packing. Nevertheless, it shows mobility /flexibility of this SrtD region. We think that our structural data and mutagenesis of sortases will help to elucidate differences in sortase pairs and will help understand their biological purpose.

Enterococcus faecalis is a multi-drug-resistant human pathogen that is found in a variety of environments and is challenging to treat. Under stress conditions, some bacteria regulate intracellular polyamine concentrations via polyamine acetyltransferases to reduce their toxicity. The E. faecalis genome encodes two polyamine acetyltransferases: PmvE and BltD. Both of these proteins belong to the Gcn5-related N-acetyltransferase (GNAT) superfamily. It is unclear why there are two enzymes with similar substrate specificities in this organism. To better understand the structure/function relationship of the E. faecalis BltD enzyme, we determined its crystal structure and performed additional assays to explore its oligomeric state and enzymatic activity. The goal was to determine whether there were structural or catalytic differences between this enzyme and other polyamine acetyltransferases that could explain this redundancy and be exploited for future development of targeted inhibitors for this important human pathogen. We found the BltD enzyme was structurally unique due to its N-terminal domain swapped dimer. However, this enzyme adopts a catalytically active monomer rather than dimer in solution . This indicates the crystal structure we obtained may represent a state that forms at high protein and salt concentrations and at low pH used during crystallization. The BltD dimer found in the crystal may represent a unique view of how an inhibitory peptide or molecule could be designed to occupy its active site. Additionally, this structure shows the extensive flexibility of the N-terminal portion of the E. faecalis BltD enzyme.

Molecular machines from the AAA+ (ATPases Associated with diverse cellular Activity) superfamily of protein disaggregases play important roles in protein folding, disaggregation and DNA processing. Recent cryo-EM structures of AAA+ molecular machines have uncovered nuanced changes in their conformation that underlie their specialized functions. Structural knowledge of these molecular machines in complex with substrates begins to explain their mechanism of activity. Here, we explore how cross-linking mass spectrometry (XL-MS) can be used to interpret changes in conformation induced by ATP in Hsp104 and how a substrate may interact with Hsp104. We applied a panel of cross-linking reagents to produce cross-linking maps of Hsp104 and interpret our data on previously determined X-ray and cryo-EM structures of Hsp104 from a thermophilic yeast, Calcarisporiella thermophila. We developed an analysis pipeline to differentiate between intra-subunit and inter-subunit contacts within the hexameric homo-oligomer. We identify cross-links that break the asymmetry that is present in Hsp104 in an ATP-hydrolysis competent conformation but is absent in an ATP-hydrolysis-defective mutant. Finally, we identify contacts between Hsp104 and a selected protein (proprotein convertase subtilisin/kexin type 9 PCSK9) to reveal contacts on the central channel of Hsp104 across the length of this protein indicating that we might have trapped interactions consistent with its translocation. Our simple and robust XL-MS-based experiments and methods help interpret how these molecular machines change conformation and bind to other proteins even in the context of homo-oligomeric assemblies enabling coupling state-of-the-art modeling approaches with XL-MS.

Serine-aspartate repeat-containing protein D (SdrD) is a cell wall-anchored, calcium-binding protein of Staphylococcus aureus. It is a member of Sdr subfamily of the microbial surface components recognizing adhesive matrix molecule (MSCRAMM) family. SdrD plays a crucial role in bacterial adhesion and pathogenesis, contributing to a wide range of infectious diseases, including skin and soft tissue infections, in both healthcare facilities and community settings. Although several Sdr structures, including complexes with peptides, have been determined, the substrate and substrate binding mechanism of SdrD remain elusive.Recently, we have determined a new crystal structure of SdrD and measured its solution small-angle X-ray scattering (SAXS). Structural analysis and comparison with existing Sdr structures as well as solution structural modelling have enhanced our understanding of this bacterial adhesin and its mechanism of molecular attachment to host cell.

Inosine 5 '-monophosphate dehydrogenase (IMPDH) is a promising antibiotic target. This enzyme catalyzes the NAD-dependent oxidation of inosine 5 '-monophosphate (IMP) to xanthosine 5 '-monophosphate (XMP), which is the rate-limiting step in guanine nucleotide biosynthesis. Bacterial IMPDH-specific inhibitors have been developed that bind to the NAD+ site. These inhibitors display varied affinities to different bacterial IMPDHs that are not easily rationalized by X-ray crystal structures of enzyme-inhibitor complexes. Inspection of X-ray crystal structures of 25 enzyme-inhibitor complexes, including 10 newly described, suggested that a mobile active site flap may be a structural determinant of inhibitor potency. Saturation transfer difference NMR experiments also suggested that the flap may contact the inhibitors to varying extents in different IMPDHs. Flap residue Leu413 contacted some inhibitors but was not structured in the crystal structures of other inhibitor complexes. The substitution of Leu413 with Phe or Ala in Bacillus anthracis IMPDH had inhibitor-selective effects, suggesting residue 413 could be a structural determinant of affinity. Curiously, the Ala substitution increased the potency of most inhibitors, even those that contacted Leu413 in the crystal structures. Presteady-state and steady-state kinetics experiments showed that the Leu413Ala substitution had comparable effects on inhibitor binding to the noncovalent EIMP complex and the covalent intermediate E-XMP*, suggesting that the flap had similar interactions in both complexes. These results demonstrate that contacts do not necessarily indicate favorable interactions, and poorly structured mobile regions should not be discounted when assessing binding determinants.

Enediynes are among the most potent antitumor and antibacterial natural products. Studies on their biosynthetic pathways have identified a shared, linear polyene precursor generated from an iterative type I polyketide synthase (PKSE) as the source of the enediyne warhead. A key step is the release of this polyene from the PKSE by a discrete thioesterase (TE). Here, we used X-ray crystallography, site-directed mutagenesis, and heterologous coexpression of PKSEs and TEs to elucidate how enediyne TEs mediate the production of the polyene. We solved the structure of wild-type EspE7 from esperamicin producer Actinomodura verrucosospora. The substrate binding pocket was also defined upon serendipitous cocrystallization of an EspE7 mutant with a fatty acyl-CoA ligand. Structural data and in vitro activity assays with EspE7 mutants provide strong evidence that Glu68 in EspE7 and the analogous Glu residue in other enediyne TEs functions as a key catalytic residue, thus supporting a hydrolysis mechanism for enediyne TEs that aligns with that of Pseudomonas sp. 4-HB-CoA TE. Furthermore, combinations of 9- and 10-membered enediyne PKSEs and TEs produced 1,3,5,7,9,11,13-pentadecaheptaene (1) as the major product. Thus, the data further support previous conclusions that 1 serves as the sole precursor for the biosynthesis of all enediyne cores.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mt), is one of the deadliest infectious diseases. The rise of multidrug-resistant strains represents a major public health threat, requiring new therapeutic options. Bacterial aminoacyl-tRNA synthetases (aaRS) have been shown to be highly promising drug targets, including for TB treatment. These enzymes play an essential role in translating the DNA gene code into protein sequence by attaching specific amino acid to their cognate tRNAs. They have multiple binding sites that can be targeted for inhibitor discovery: amino acid binding pocket, ATP binding pocket, tRNA binding site and an editing domain. Recently we reported several high-resolution structures of M. tuberculosis phenylalanyl-tRNA synthetase (MtPheRS) complexed with tRNAPhe and either L-Phe or a nonhydrolyzable phenylalanine adenylate analog. Here, using Nucleic Magnetic Resonance (NMR) and Surface Plasmon Resonance (SPR) we identified fragments that bind to MtPheRS and we determined crystal structures of their complexes with MtPheRS/tRNAPhe. All the binders interact with the L-Phe amino acid binding site. The analysis of interactions of the new compounds combined with adenylate analog structure provides insights for the rational design of anti-tuberculosis drugs. The 3’ arm of the tRNAPhe in all the structures was disordered with exception of one complex with D-735 compound. In this structure the 3’ CCA end of the acceptor stem is observed in the editing domain of MtPheRS providing insights regarding the post-transfer editing activity of class II aaRS.

Tuberculosis (TB) is recognized as the second leading cause of death globally from a single infectious agent, following SARS-CoV-2 pneumonia. Aminoacyl-tRNA synthetases (aaRS) are essential enzymes responsible for attaching amino acids to their cognate tRNAs. These enzymes represent a promising set of targets for selective drug design due to the divergence between prokaryotic and eukaryotic aaRS. Recently, a new class of 3-aminopyrazine- 2-carboxamide derivatives has been identified as potent inhibitors of prolyl-tRNA synthetase (ProRS) from Mycobacterium tuberculosis (Mtb). These compounds exhibit significant antimycobacterial activity against Multi Drug Resistant strains of Mtb and demonstrate cytotoxicity against HepG2 human hepatocellular carcinoma cells. In this study, we present the crystal structures of MtbProRS in complex with five distinct 3-aminopyrazine-2-carboxamide derivatives. Structural analysis reveals that these inhibitors compete with ATP for the binding site of MtbProRS. Importantly, a critical hydrogen bond between the Glu144 of MtbProRS and the amino group of the carboxamide is identified, providing insights into the selective inhibition of MtbProRS over human ProRS.

Spermidine/spermine N-acetyltransferases (SSATs) and other types of polyamine acetyltransferases (PAATs) acetylate diamines and/or polyamines. These enzymes are evolutionarily related and belong to the Gcn5-related N-acetyltransferase (GNAT) superfamily, yet we lack a fundamental understanding of their substrate specificity and/or promiscuity toward different compounds. Many of these enzymes are known or are predicted to acetylate polyamines, but in the cell there are other types of compounds that contain moieties derived from polyamines that may be the native substrates for these enzymes. To learn more about the identity of substrates that are acetylated, we selected and screened 17 different GNAT enzymes for activity toward a set of structurally diverse compounds that contained different types of amine moieties (e.g., aminopropyl, aminobutyl, etc.). These compounds included diamines, triamines, and polyamines containing primary amino groups, and they had structural diversity with variation of the chain length and presence or absence of internal amino groups and other functional groups. We found 12 of the 17 enzymes acetylated at least one of the compounds. Some enzymes were selective toward acetylating only one compound while others exhibited substrate promiscuity toward numerous compounds. Our experimental results ultimately allowed us to pinpoint specific substrates that could be further investigated to more fully understand substrate specificity versus promiscuity of GNAT enzymes and the role of acetylated small molecules in cells.

Ultra-high-resolution crystal structures of proteins provide critical insights into protein structure, dynamics, hydrogen bonding, and solvent networks. Crambin, a small hydrophobic storage protein consisting of 46 residues (4.7 kDa), is found in the embryonic tissue of seeds from Crambe abyssinica. This protein is renowned for its ability to crystallize readily, forming some of the best-ordered macromolecular crystals known, which diffract X-rays to the highest sub-atomic resolution recorded for any protein to date.We have previously reported the room temperature structure of crambin, refined to an exceptional resolution of 0.70 Å using SHELXL. That analysis revealed intricate details of the dynamic solvent network, characterized by alternative side chain conformations and shifts in water molecule positions. In this work, we extend our investigation by presenting new structural data collected at cryogenic temperatures: 15K using liquid helium and 100K using liquid nitrogen cooling.We will report the ultra-high-resolution structures at 15K and 100K, providing a comparative analysis of the solvent networks across these different temperature datasets. This comparison aims to deepen our understanding of the solvent and protein dynamics, offering valuable insights into the protein interactions within solvent environments. Our findings underscore the significance of ultrahigh-resolution crystallography in elucidating the complex interplay between proteins and their solvent environments, with potential implications for the broader field of structural biology.

Hydration plays critical role in protein structure, stability, dynamics and function. Recent advancements in high-resolution crystallography have provided a clearer understanding of the hydration patterns in proteins and their complexes with nucleic acids. The high level of detail allows to observe hydration patterns that were previously unresolved. Water-mediated specific protein interaction with DNA were initially discovered for E. coli the tryptophan repressor (TrpR) operator complex. Fixed water molecules are strategically positioned to mediate interactions between the repressor and the DNA bases. Here we report determination of high- resolution structures of TrpR and its complexes from Francisella tularensis including: aporepressor, complexes with L-Trp (activator), indole propionic acid (IPA) (inhibitor) and with specific F. tularensis trp operator. Structure analysis shows conservation of ligand binding and specific repressor-DNA interactions, including water-mediated and suggests that the majority of these interactions may be preserved across TrpR family. This research highlights the crucial role that water molecules play in mediating interactions between the repressor protein and the DNA bases that define the operator's identity. Bound water molecules are likely underrepresented in models of the interface of many specific biological complexes either because of spatial disorder in the crystal or inadequate resolution. These water molecules are not just passive participants; they are actively involved in the stereospecific recognition process, forming a dynamic and reversible network that extends the functional surfaces of the macromolecules involved. While this study focuses on the TrpR/operator system, the findings may have broader implications for understanding other protein-nucleic acid interactions and macromolecular complexes.

Coronavirus nucleocapsid protein (NP) of SARS-CoV-2 plays a central role in many functions important for virus proliferation including packaging and protecting genomic RNA. The protein shares sequence, structure, and architecture with nucleocapsid proteins from betacoronaviruses. The N-terminal domain (NPRBD) binds RNA and the C-terminal domain is responsible for dimerization. After infection, NP is highly expressed and triggers robust host immune response. The anti-NP antibodies are not protective and not neutralizing but can effectively detect viral proliferation soon after infection. Two structures of SARS-CoV-2 NPRBD were determined providing a continuous model from residue 48 to 173, including RNA binding region and key epitopes. Five structures of NPRBD complexes with human mAbs were isolated using an antigen-bait sorting. Complexes revealed a distinct complement-determining regions and unique sets of epitope recognition. This may assist in the early detection of pathogens and designing peptide-based vaccines. Mutations that significantly increase viral load were mapped on developed, full length NP model, likely impacting interactions with host proteins and viral RNA.

Ultrahigh-resolution structures provide unprecedented details about protein dynamics, hydrogen bonding and solvent networks. The reported 0.70 Å, room-temperature crystal structure of crambin is the highest-resolution ambient-temperature structure of a protein achieved to date. Sufficient data were collected to enable unrestrained refinement of the protein and associated solvent networks using SHELXL. Dynamic solvent networks resulting from alternative side-chain conformations and shifts in water positions are revealed, demonstrating that polypeptide flexibility and formation of clathrate-type structures at hydrophobic surfaces are the key features endowing crambin crystals with extraordinary diffraction power.