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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.

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.

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.

Research has been conducted on Mytilus edulis PEP mutase. The crystal structure of this mutase in complex with the substrate-analogue inhibitor has been investigated and the results indicate that the phosphoryl group transfer occurs on PEP Si-face with the configuration retention at phosphorus.

Structural analysis of the enzyme-substrate complex reveals that phosphonopyruvate binds in the alpha/beta-barrel's central channel of the Beta-strands C-termini. The structure resembles that of pyruvate kinase but differes in the acid/base catalysis function.

Research on a mutant form of beta lactamase from Staphylococcus aureus shows that substrate specificity is not determined at the acyl-enzyme state. This was the conclusion of researchers who studied the binding modes of benzylpenicillin and cephaloridine.

The mechanism of phosphoenolpyruvate mutase (PEP mutase) catalysis is discussed relative to site-directed mutagenesis studies of active site residues. The sequencing, expression, cloning, and mutagenesis of the Mytilus edulis PEP mutase gene in Escherichia coli are covered. A catalytic motif has been proposed. It could serve for both phosphoryl transfer and the C--C cleavage enzymes of the PEP mutase superfamily.

Research was conducted to identify the specificity requirements for phosphorylation of Mycoplasma capricolum HPr. A mutagenesis method was used in which residues 43-57 in a chimera were replaced by the Escherichia coli sequence. Results indicate that the kinase-HPr interaction takes place mostly through a hydrophobic region. Comparisons conducted between the chimeric construct and M. capricolum provide a basis for the interpretation of activity assays results.

The effect of the omega-loop deletion in the function and structure of the enzyme beta-lactamases was investigated. A beta-lactamase mutant protein was produced using the four-primer overlap-extension method. A double-stranded plasmid DNA was then used to resequence the beta-lactamase gene. It was found that the absence of the omega-loop eliminated Glu166, a primary catalytic residue, thus resulting in dysfunctional deacylation. It also eliminated the enzyme's ability to hydrolyze beta-lactam compounds.

The presence of a non-proline cis peptide between Glu166 and Ile 167 in the active site of beta-lactamase from Staphylococcus aureaus PC1 was determined. A site-directed mutant N136A was produced to examine the role of the interaction between the side chain of Asn136 and the main chain of Glu166. No measurable hydrolytic activity towards penicillins or cephalosporis by the enzyme was observed, apart from the chromegenic cephalosporin, nitrocefin. The hydrolysis of nitrocefin after reincubation with several beta-lactam compounds was monitored, and cephalosporins were found to form stable acyl complexes with the enzyme.Electrospray mass spectrometry was used to determine the molecular weight of the mutant.

The structure and enzymatic functions of beta-lactamase from Staphylococcus aureus PC1 was evaluated through construction of two circularly permuted proteins, cp228 and cp254. Both have an eight residue long peptide that joins the N- and C- terminal helices of the native molecule with N-terminal residue located on positions 228 and 254, respectively. Cp228 has shown a decrease of activity towards beta-lactam antibiotics and a greater activity towards cefataxime.

Two site-directed mutant enzymes of the class A beta-lactamase from Staphylococcus aureus PC1 were produced to block the site occupied by a hydrolytic water molecule in the native enzyme. The crystal structures of these two mutant enzymes, N170Q and N170M, were determined. Results indicated that the water site is preserved and that Met170 occupies part of the space that is needed for substrate binding. The short contacts between the substrate and the enzyme may cause structure perturbation and inactivation.

Two mutant beta-lactamases from Staphylococcus aureus PC1 which probe key catalytic residues were formed by site-directed mutagenesis. The nucleophilic group that attacks the beta-lactam carbonyl carbon atom was removed in the S70A enzyme. The crystal structure of S70A beta-lactamase was determined at 2.1 angstrom resolution. The structure is similar to that of the native enzyme except for the mutation site. The K73H beta-lactamase was tested for the possible role of Lys73 in proton transfer. It did not show any activity toward benzylpenicillin. The crystal structure of K73H beta-lactamase was determined at 1.9 angstrom resolution.

Photoaffinity labeling, site-directed mutagenesis and structural analysis of pyruvate phosphate dikinase from Clostridium symbiosum reveals that the nucleotide binding site is present in the N-terminal. X-ray crystal structure studies indicates that the enzyme folds into N-terminal, central and C-terminal regions. ATP binds in a crevice formed by subdomains one and three in a manner similar to ADP binding in D-alanine-D-alanine-ligase.

An unexpected structural link to legume lectins, notwithstanding the absence of sequence homology, is evident in the crystal structure of a 14-kDa bovine spleen S-lectin complexed with the disaccharide N-acetyllactosamine at a resolution of 1.9 angstroms. An extended beta-sandwich results from the association of a pair of monomers. Complex interactions that limit the position of the O4 atom are responsible for the specificity of beta-galactose.

A mutated form of troponin c from chicken skeletal muscle was used as a spectral probe for monitoring the Ca2+-induced structrural transition of the regulatory N domain. Phe-29 at the COOH-terminal fragment of the A helix near the Ca2+-binding loop of site I was replaced by tryptophan (trp) which elicited unambiguous spectral features owing to the absence of other trp or tyrosine. Alteration in spectral features was restricted to Ca2+-binding in the N domain conformational transition. The spectral feature of the probe mutant was also applied to the analysis of Ca2+-binding affinities of five double mutants wherein nonpolar amino acids were replaced by polar residues.

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.