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p97 is a hexameric AAA+ adenosine triphosphatase (ATPase) that is an attractive target for cancer drug development. We report cryo-electron microscopy (cryo-EM) structures for adenosine diphosphate (ADP)-bound, full-length, hexameric wild-type p97 in the presence and absence of an allosteric inhibitor at resolutions of 2.3 and 2.4 angstroms, respectively. We also report cryo-EM structures (at resolutions of ~3.3, 3.2, and 3.3 angstroms, respectively) for three distinct, coexisting functional states of p97 with occupancies of zero, one, or two molecules of adenosine 5'-O-(3-thiotriphosphate) (ATPγS) per protomer. A large corkscrew-like change in molecular architecture, coupled with upward displacement of the N-terminal domain, is observed only when ATPγS is bound to both the D1 and D2 domains of the protomer. These cryo-EM structures establish the sequence of nucleotide-driven structural changes in p97 at atomic resolution. They also enable elucidation of the binding mode of an allosteric small-molecule inhibitor to p97 and illustrate how inhibitor binding at the interface between the D1 and D2 domains prevents propagation of the conformational changes necessary for p97 function.

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.

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.

The activation of mammalian glutamate dehydrogenase occurs by ADP opening the catalytic cleft by binding to the NADH site. Results indicate that the antenna structure in the enzyme evolves before the formation of purine regulatory sites and that the enzyme acquires allosteric regulation function as it evolves.

We report the solution structure of Escherichia coli β-galactosidase (∼465 kDa), solved at ∼3.2-Å resolution by using single-particle cryo-electron microscopy (cryo-EM). Densities for most side chains, including those of residues in the active site, and a catalytic Mg2+ ion can be discerned in the map obtained by cryo-EM. The atomic model derived from our cryo-EM analysis closely matches the 1.7-A crystal structure with a global rmsd of ∼0.66 Å. There are significant local differences throughout the protein, with clear evidence for conformational changes resulting from contact zones in the crystal lattice. Inspection of the map reveals that although densities for residues with positively charged and neutral side chains are well resolved, systematically weaker densities are observed for residues with negatively charged side chains. We show that the weaker densities for negatively charged residues arise from their greater sensitivity to radiation damage from electron irradiation as determined by comparison of density maps obtained by using electron doses ranging from 10 to 30 e-2∼/Å2. In summary, we establish that it is feasible to use cryo-EM to determine near-atomic resolution structures of protein complexes (<500 kDa) with low symmetry, and that the residue-specific radiation damage that occurs with increasing electron dose can be monitored by using dose fractionation tools available with direct electron detector technology. [ABSTRACT FROM AUTHOR]

Summary: The Bcl-2 family member Bax translocates from the cytosol to mitochondria, where it oligomerizes and permeabilizes the mitochondrial outer membrane to promote apoptosis. Bax activity is counteracted by prosurvival Bcl-2 proteins, but how they inhibit Bax remains controversial because they neither colocalize nor form stable complexes with Bax. We constrained Bax in its native cytosolic conformation within cells using intramolecular disulfide tethers. Bax tethers disrupt interaction with Bcl-xL in detergents and cell-free MOMP activity but unexpectedly induce Bax accumulation on mitochondria. Fluorescence loss in photobleaching (FLIP) reveals constant retrotranslocation of WT Bax, but not tethered Bax, from the mitochondria into the cytoplasm of healthy cells. Bax retrotranslocation depends on prosurvival Bcl-2 family proteins, and inhibition of retrotranslocation correlates with Bax accumulation on the mitochondria. We propose that Bcl-xL inhibits and maintains Bax in the cytosol by constant retrotranslocation of mitochondrial Bax. [ABSTRACT FROM AUTHOR]

Looks at a study which examined the structure of the class A cap beta-lactamases with reference to its relation with cap omega-loop. How the crystal structure was determined; Methodology used to conduct the study; Results of the study.

The core of skeletal muscle Z-discs consists of actin filaments from adjacent sarcomeres that are cross-linked by α-actinin homodimers. Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP)/Cypher interacts with α-actinin, myotilin, and other Z-disc proteins via the PDZ domain. However, these interactions are not sufficient to maintain the Z-disc structure. We show that ZASP directly interacts with skeletal actin filaments. The actin-binding domain is between the modular PDZ and LIM domains. This ZASP region is alternatively spliced so that each isoform has unique actin-binding domains. All ZASP isoforms contain the exon 6-encoded ZASP-like motif that is mutated in zaspopathy, a myofibrillar myopathy (MFM), whereas the exon 8-11 junction-encoded peptide is exclusive to the postnatal long ZASP isoform (ZASP-LΔex10). MFM is characterized by disruption of skeletal muscle Z-discs and accumulation of myofibrillar degradation products. Wild-type and mutant ZASP interact with α-actin, α-actinin, and myotilin. Expression of mutant, but not wild-type, ZASP leads to Z-disc disruption and F-actin accumulation in mouse skeletal muscle, as in MFM. Mutations in the actin-binding domain of ZASP-LΔex10, but not other isoforms, cause disruption of the actin cytoskeleton in muscle cells. These isoform-specific mutation effects highlight the essential role of the ZASP-LΔex10 isoform in F-actin organization. Our results show that MFM-associated ZASP mutations in the actin-binding domain have deleterious effects on the core structure of the Z-discs in skeletal muscle. [ABSTRACT FROM AUTHOR]