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The CASP14 experiment demonstrated the extraordinary structure modeling capabilities of artificial intelligence (AI) methods. That result has ignited a fierce debate about what these methods are actually doing. One of the criticisms has been that the AI does not have any sense of the underlying physics but is merely performing pattern recognition. Here, we address that issue by analyzing the extent to which the methods identify rare structural motifs. The rationale underlying the approach is that a pattern recognition machine tends to choose the more frequently occurring motifs, whereas some sense of subtle energetic factors is required to choose infrequently occurring ones. To reduce the possibility of bias from related experimental structures and to minimize the effect of experimental errors, we examined only CASP14 target protein crystal structures determined to a resolution limit better than 2 Å, which lacked significant amino acid sequence homology to proteins of known structure. In those experimental structures and in the corresponding models, we track cis peptides, π- helices, 3 10- helices, and other small 3D motifs that occur in the PDB database at a frequency of lower than 1% of total amino acid residues. The best-performing AI method, AlphaFold2, captured these uncommon structural elements exquisitely well. All discrepancies appeared to be a con- sequence of crystal environment effects. We propose that the neural network learned a protein structure potential of mean force, enabling it to correctly identify situations where unusual structural features represent the lowest local free energy because of subtle influences from the atomic environment. [ABSTRACT FROM AUTHOR]

The crystal structure studies of a second conformational state of Clostridium symbiosum pyruvate phosphate dikinase (PPDK) are reported. The studies confirm the swivel motion of the His domain adjacent to the phosphoenolpyruvate (PEP)-binding domain.

Human hyaluronidase-1 is implicated in cancer proliferation, angiogenesis and inflammatory diseases. Its expression is up regulated in advanced stages of bladder cancer, whereas the expression of the alternative splice variants is down regulated.

Bacterial transport of many sugars, coupled to their phosphorylation, is carried out by the phosphoenolpyruvate (PEP): sugar phosphotransferase system and involves five phosphoryl group transfer reactions. Sugar translocation initiates with the [Mg.sup.2+]-dependent phosphorylation of enzyme I (El) by PEP. Crystals of Escherichia coli El were obtained by mixing the protein with [Mg.sup.2+] and PEP, followed by oxalate, an El inhibitor. The crystal structure reveals a dimeric protein where each subunit comprises three domains: a domain that binds the partner PEP:sugar phosphotransferase system protein, HPr; a domain that carries the phosphorylated histidine residue, His-189; and a PEP-binding domain. The PEP-binding site is occupied by [Mg.sup.2+] and oxalate, and the phosphorylated His-189 is in-line for phosphotransfer to/from the ligand. Thus, the structure represents an enzyme intermediate just after phosphotransfer from PEP and before a conformational transition that brings His-189~P in proximity to the phosphoryl group acceptor, His-15 of HPr. A model of this conformational transition is proposed whereby swiveling around an [alpha]-helical linker disengages the His domain from the PEP-binding domain. Assuming that HPr binds to the HPr-binding domain as observed by NMR spectroscopy of an El fragment, a rotation around two linker segments orients the His domain relative to the HPr-binding domain so that His-189~P and His-15 are appropriately stationed for an in-line phosphotransfer reaction. sugar transport | phosphorylation | x-ray crystallography

Contributions made by Pseudomonas aeruginosa (PaADI) core residues Cys406, His278, and Asp166 and the contribution from Asp280 to catalysis of the formation and hydrolysis of the Cys406-alkyluronium intermediate were assessed by kinetic analysis of site-directed mutants. The results suggest that electrostatic interactions play a dominant role in PaADI catalysis.

Study was conducted to define the chemical function, a new member of the isocitrate lyase enzyme family derived from the flowering plant Dianthus caryophyllus. A substrate screen that employed a (2R)-malate core structure was designed on the basis of an analysis of the structural contexts of sequence markers common to the C-C bond lyases of the isocitrate lyase/phosphoenolpyruvate mutase superfamily.

Expression of PSR132 protein from Dianthus caryophyllus was induced in response to ethylene production that was associated with petal senescence and therefore the protein is named petal death protein (PDP). The crystal structure of PDP was determined at 2.7 angstrom resolution, indicating that a dimer-of-dimers oligomeric association.

The ygJZ gene product of Escherichia coli represents a large protein family conserved in bacteria to eukaryotes. The members of this family are uncharacterized proteins with marginal sequence similarity to the T-protein (aminomethyltransferase) of the glycine cleavage system. To assist with the functional assignment of the YgfZ family, the crystal structure of the E. coli protein was determined by multiwavelength anomalous diffraction. The protein molecule has a three-domain architecture with a central hydrophobic channel. The structure is very similar to that of bacterial dimethylglycine oxidase, an enzyme of the glycine betaine pathway and a homolog of the T-protein. Based on structural superposition, a folate-binding site was identified in the central channel of YgfZ, and the ability of YgfZ to bind folate derivatives was confirmed experimentally. However, in contrast to dimethylglycine oxidase and T-protein, the YgfZ family lacks amino acid conservation at the folate site, which implies that YgfZ is not an aminomethyltransferase but is likely a folate-dependent regulatory protein involved in one-carbon metabolism.

Critical assessment of structure prediction (CASP) conducts community experiments to determine the state of the art in computing protein structure from amino acid sequence. The process relies on the experimental community providing information about not yet public or about to be solved structures, for use as targets. For some targets, the experimental structure is not solved in time for use in CASP. Calculated structure accuracy improved dramatically in this round, implying that models should now be much more useful for resolving many sorts of experimental difficulties. To test this, selected models for seven unsolved targets were provided to the experimental groups. These models were from the AlphaFold2 group, who overall submitted the most accurate predictions in CASP14. Four targets were solved with the aid of the models, and, additionally, the structure of an already solved target was improved. An a posteriori analysis showed that, in some cases, models from other groups would also be effective. This paper provides accounts of the successful application of models to structure determination, including molecular replacement for X‐ray crystallography, backbone tracing and sequence positioning in a cryo‐electron microscopy structure, and correction of local features. The results suggest that, in future, there will be greatly increased synergy between computational and experimental approaches to structure determination. [ABSTRACT FROM AUTHOR]

The crystal structure determination of recombinant GK[alpha][beta] and GK[beta][beta] from the marine worm Namalycastis sp. with a transition state analogue, [Mg.sup.2+]-ADP-N[O.sup.3-]-glycocyamine is described. The active site environments of the glycocyamine kinase (GK), homodimeric creatine kinase (CK), and monomeric arginine kinase (AK) at the bound states provide insight into the structural determinants of substrate specificity and the coupling between the structural asymmetry and negative cooperativity proposed for CK which is not supported in the case of GK.

Several studies are conducted to explain the substrate binding and stereoselectivity phenomena observed in the Giardia lamblia fructose-1,6-bisphosphate aldolase (FBPA), a member of the II zinc-dependent aldolase family. The polarization of the substrate for protein transfer in the FBPA compound is shown to be highly dependent on the zinc mobility within it.

The biochemical characterization and cystallographic analysis of HI0827 protein isolated from Haemophilus influenza Rd KW20 was carried out to examine the structural variations within the acyl-CoA thioesterase family. The findings provided insight into a new structural class of hexameric hot dog fold enzyme, acyl-coenzyme A (acyl-CoA) thioesterases.

A study was conducted to examine the biochemical and biological function of the hot dog fold thioesterase YciA (EcYciA) from Escherichia coli and its close sequence homologue HI0827 from Haemophilus influenzae (HiYciA). The results provide insight into significant role of YciA for efficient CoA-regulated hydrolysis of cellular acyl-CoA thioesters in a wide range of bacteria.

The isolation, structure and chemical mechanism for catalysis of the Pseudomonas aeruginosa, arginine deiminase (ADI) (L-arginine) which involves initial formation and subsequent hydrolysis of a Cys-alkylthiouronium ion intermediate is described. The findings from the studies would help to design the arginine analogs such as L-canavanine that migaht react with the ADIs to form inactive covalent adduct during catalytic turnover.

The crystal structure of PA4872 from Pseudomonas aeruginosa in complex with oxalate and [Mg.sup.2+] is studied. The results have shown that PA4872 is an oxaloacetate decarboxylase, which is used primarily by pseudomonas, and that the enzyme is used by differential bacterial species to maintain pyruvate and bicarbonate cellular levels in many pathways.

The structure and properties of recombinant phosphonopyruvate (P-pyr) hydrolase (PPH) from variovorax sp. Pal2 expressed in E. coli is reported. Structure alignment of PPH with other superfamily members revealed two pairs of invariant or conservatively replaced residues that anchor the flexible gating loop.

The two crystal structures of the C123S mutant of 2-methylisocitrate lyase have been determined, one with the bound reaction products. The structure-based sequence analysis and phylogenic tree construction reveal determinants of substrate specificity, highlight nodes of divergence of families, and predict enzyme families with new functions.

The results from transient-state kinetic experiments which show that arginine deiminase catalysis proceeds through a covalent adduct formed by activesite Cys406 displacement of ammonia from the guanidium carbon of L-arginine are presented.

The crystal structures of wild-type and D58A mutases, in the apo state and in complex with Mg(II), are presented. The members of the phosphoenolpyruvate (PEP) mutase/isocitratelyase (ICL) family share sequence homology and overall fold, and use similar modes of flexibility.

Abstract: The functional and biological significance of the selected CASP12 targets are described by the authors of the structures. The crystallographers discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP12 experiment. [ABSTRACT FROM AUTHOR]

Bacterial Resistance to Β-Lactam Antibiotics: Crystal Structure of Β-Lactamase from Staphylococcus aureus PCI at 2.5 Å Resolution MANY TYPES OF DEFENSE MECHANISMS EXIST IN THEbiologigal world, and relatively lowly life [...]

Bacteriophage tailspike proteins act as primary receptors, often possessing endoglycosidase activity toward bacterial lipopolysaccharides or other exopolysaccharides, which enable phage absorption and subsequent DNA injection into the host. Phage CBA120, a contractile long-tailed Viunalikevirus phage infects the virulent Escherichia coli O157:H7. This phage encodes four putative tailspike proteins exhibiting little amino acid sequence identity, whose biological roles and substrate specificities are unknown. Here we focus on the first tailspike, TSP1, encoded by the orf210 gene. We have discovered that TSP1 is resistant to protease degradation, exhibits high thermal stability, but does not cleave the O157 antigen. An immune-dot blot has shown that TSP1 binds strongly to non-O157:H7 E. coli cells and more weakly to K. pneumoniae cells, but exhibits little binding to E. coli O157:H7 strains. To facilitate structure-function studies, we have determined the crystal structure of TSP1 to a resolution limit of 1.8 Å. Similar to other tailspikes proteins, TSP1 assembles into elongated homotrimers. The receptor binding region of each subunit adopts a right-handed parallel β helix, reminiscent yet not identical to several known tailspike structures. The structure of the N-terminal domain that binds to the virion particle has not been seen previously. Potential endoglycosidase catalytic sites at the three subunit interfaces contain two adjacent glutamic acids, unlike any catalytic machinery observed in other tailspikes. To identify potential sugar binding sites, the crystal structures of TSP1 in complexes with glucose, α-maltose, or α-lactose were determined. These structures revealed that each sugar binds in a different location and none of the environments appears consistent with an endoglycosidase catalytic site. Such sites may serve to bind sugar units of a yet to be identified bacterial exopolysaccharide. [ABSTRACT FROM AUTHOR]

For the last two decades, CASP has assessed the state of the art in techniques for protein structure prediction and identified areas which required further development. CASP would not have been possible without the prediction targets provided by the experimental structural biology community. In the latest experiment, CASP10, more than 100 structures were suggested as prediction targets, some of which appeared to be extraordinarily difficult for modeling. In this article, authors of some of the most challenging targets discuss which specific scientific question motivated the experimental structure determination of the target protein, which structural features were especially interesting from a structural or functional perspective, and to what extent these features were correctly reproduced in the predictions submitted to CASP10. Specifically, the following targets will be presented: the acid-gated urea channel, a difficult to predict transmembrane protein from the important human pathogen Helicobacter pylori; the structure of human interleukin (IL)−34, a recently discovered helical cytokine; the structure of a functionally uncharacterized enzyme OrfY from Thermoproteus tenax formed by a gene duplication and a novel fold; an ORFan domain of mimivirus sulfhydryl oxidase R596; the fiber protein gene product 17 from bacteriophage T7; the bacteriophage CBA-120 tailspike protein; a virus coat protein from metagenomic samples of the marine environment; and finally, an unprecedented class of structure prediction targets based on engineered disulfide-rich small proteins. Proteins 2014; 82(Suppl 2):26-42. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The parasite Giardia lamblia utilizes the L-arginine dihydrolase pathway to generate ATP from L-arginine. Carbamate kinase (CK) catalyzes the last step in this pathway, converting ADP and carbamoyl phosphate to ATP and ammonium carbamate. Because the L-arginine pathway is essential for G. lamblia survival and absent in high eukaryotes including humans, the enzyme is a potential target for drug development. We have determined two crystal structures of G. lamblia CK (glCK) with bound ligands. One structure, in complex with a nonhydrolyzable ATP analog, adenosine 5′-adenylyl-β,γ-imidodiphosphate (AMP-PNP), was determined at 2.6 Å resolution. The second structure, in complex with citric acid bound in the postulated carbamoyl phosphate binding site, was determined in two slightly different states at 2.1 and 2.4 Å resolution. These structures reveal conformational flexibility of an auxiliary domain (amino acid residues 123–170), which exhibits open or closed conformations or structural disorder, depending on the bound ligand. The structures also reveal a smaller conformational change in a region associated the AMP-PNP adenine binding site. The protein residues involved in binding, together with a model of the transition state, suggest that catalysis follows an in-line, predominantly dissociative, phosphotransfer reaction mechanism, and that closure of the flexible auxiliary domain is required to protect the transition state from bulk solvent. [ABSTRACT FROM AUTHOR]

Human RON (Recepteur d'Origine Nantais) receptor tyrosine kinase is a cell surface receptor for Macrophage Stimulating Protein (MSP). RON mediates signal transduction pathways that regulate cell adhesion, invasion, motility and apoptosis processes. Elevated levels of RON and its alternatively spliced variants are implicated in the progression and metastasis of tumor cells. The binding of MSP α/β heterodimer to the extracellular region of RON receptor induces receptor dimerization and activation by autophosphorylation of the intracellular kinase domains. The ectodomain of RON, containing the ligand recognition and dimerization domains, is composed of a semaphorin (Sema), Plexins-Semaphorins-Integrins domain (PSI), and four Immunoglobulins-Plexins-Transcription factor (IPT) domains. High affinity association between MSP and RON is mediated by the interaction between MSP β-chain and RON Sema, although RON activation requires intact RON and MSP proteins. Here, we report the structure of RON Sema-PSI domains at 1.85 Å resolution. RON Sema domain adopts a sevenbladed β-propeller fold, followed by disulfide bond rich, cysteine-knot PSI motif. Comparison with the homologous Met receptor tyrosine kinase reveals that RON Sema-PSI contains distinguishing secondary structural features. These define the receptors' exclusive selectivity towards their respective ligands, RON for MSP and Met for HGF. The RON Sema-PSI crystal packing generates a homodimer with interface formed by the Sema domain. Mapping of the dimer interface using the RON homology to Met, MSP homology to Hepatocyte Growth Factor (HGF), and the structure of the Met/HGF complex shows the dimer interface overlapping with the putative MSPb binding site. The crystallographically determined RON Sema-PSI homodimer may represent the dimer assembly that occurs during ligand-independent receptor activation and/or the inhibition of the constitutive activity of ROND160 splice variant by the soluble RON splice variant, RONΔ85. [ABSTRACT FROM AUTHOR]

High throughput genome wide associations studies (GWAS) are now identifying a large number of genome loci related to risk of common human disease. Each such locus presents a challenge in identifying the relevant underlying mechanism. Here we report the experimental characterization of a proposed causal single nucleotide polymorphism (SNP) in a locus related to risk of Crohn's disease and ulcerative colitis. The SNP lies in the MST1 gene encoding Macrophage Stimulating Protein (MSP), and results in an R689C amino acid substitution within the β-chain of MSP (MSPβ). MSP binding to the RON receptor tyrosine kinase activates signaling pathways involved in the inflammatory response. We have purified wild-type and mutant MSPβ proteins and compared biochemical and biophysical properties that might impact the MSP/RON signaling pathway. Surface plasmon resonance (SPR) binding studies showed that MSPβ R689C affinity to RON is approximately 10-fold lower than that of the wild-type MSPβ and differential scanning fluorimetry (DSF) showed that the thermal stability of the mutant MSPβ was slightly lower than that of wild-type MSPβ, by 1.6 K. The substitution was found not to impair the specific Arg483-Val484 peptide bond cleavage by matriptase-1, required for MSP activation, and mass spectrometry of tryptic fragments of the mutated protein showed that the free thiol introduced by the R689C mutation did not form an aberrant disulfide bond. Together, the studies indicate that the missense SNP impairs MSP function by reducing its affinity to RON and perhaps through a secondary effect on in vivo concentration arising from reduced thermodynamic stability, resulting in down-regulation of the MSP/RON signaling pathway. [ABSTRACT FROM AUTHOR]

Transcription factor p63, a p53 family member, plays a role in epithelial cell development, cell cycle arrest, apoptosis, and tumorigenesis. Point mutations, primarily in the DNA binding domain (p63DBD). lead to malformation syndromes. To gain insight into differences between p63 and p53 and the impact of mutations on the structure, we have determined two crystal structures of p63DBD in complex with A/T-rich response elements. One complex contains a 10-bp DNA half-site response element (5'AAACATGT- TT3') and the other contains a 22-bp DNA full response element with a 2-bp spacer between two half-sites (5'AAACATGTTTTAAAA- CATGTTT3'). In both structures, each half-site binds a p63DBD dimer. The two p63DBD dimers do not interact in the presence of the DNA spacer, whereas they interact with one another in the p63DBD/10-bp complex where the DNA simulates a full response element by packing end-to-end. A unique dimer-dimer interaction involves a variable loop region, which differs in length and se- quence from the counterpart loop of p53DBD. The DNA trajectories in both structures assume superhelical conformations. Surface plasmon resonance studies of p63DBD/DNA binding yielded Kd = 11.7 μM for a continuous full response element, whereas binding was undetectable with the 22-bp DNA, suggesting an important contribution of a p63DBD interdimer interface to binding and establishing that p63DBD affinity to the response element is approximately 1,000-fold lower than that of p53DBD. Analyses of the structural consequences of p63DBD mutations that cause develop- mental defects show that, although some mutations affect DNA binding directly, the majority affects protein stability. [ABSTRACT FROM AUTHOR]

Carbamate kinase catalyzes the reversible conversion of carbamoyl phosphate and ADP to ATP and ammonium carbamate, which is hydrolyzed to ammonia and carbonate. The three-dimensional structure of carbamate kinase from the human parasite Giardia lamblia ( glCK) has been determined at 3 Å resolution. The crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 69.77, b = 85.41, c = 102.1 Å, β = 106.8°. The structure was refined to a final R factor of 0.227. The essentiality of glCK together with its absence in humans makes the enzyme an attractive candidate for anti- Giardia drug development. Steady-state kinetic rate constants have been determined. The kcat for ATP formation is 319 ± 9 s−1. The Km values for carbamoyl phosphate and ADP are 85 ± 6 and 70 ± 5 µ M, respectively. The structure suggests that three invariant lysine residues (Lys131, Lys216 and Lys278) may be involved in the binding of substrates and phosphoryl transfer. The structure of glCK reveals that a glycerol molecule binds in the likely carbamoyl phosphate-binding site. [ABSTRACT FROM AUTHOR]

HI1506 is a 128-residue hypothetical protein of unknown function from Haemophilus influenzae. It was originally annotated as a shorter 85-residue protein, but a more detailed sequence analysis conducted in our laboratory revealed that the full-length protein has an additional 43 residues on the C terminus, corresponding with a region initially ascribed to HI1507. As part of a larger effort to understand the functions of hypothetical proteins from Gram-negative bacteria, and H. influenzae in particular, we report here the three-dimensional solution NMR structure for the corrected full-length HI1506 protein. The structure consists of two well-defined domains, an α/β 50-residue N-domain and a 3-α 32-residue C-domain, separated by an unstructured 30-residue linker. Both domains have positively charged surface patches and weak structural homology with folds that are associated with RNA binding, suggesting a possible functional role in binding distal nucleic acid sites. [ABSTRACT FROM AUTHOR]