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Z band alternately spliced PDZ-containing protein (ZASP) is a sarcomere Z disk protein expressed in human cardiac and skeletal muscle that is thought to be involved in a dominant familial dilated cardiomyopathy. The N-terminal PDZ domain of ZASP interacts with the C terminus of α-actinin-2, the major component of the Z disk, probably by forming a ternary complex with titin Z repeats. We have determined...
Mechanisms leading to gene variations are responsible for the diversity of species and are important components of the theory of evolution. One constraint on gene evolution is that of protein foldability; the three-dimensional shapes of proteins must be thermodynamically stable. We explore the impact of this constraint and calculate properties of foldable sequences using 3660 structures from the Protein...
Protein crystallization constitutes a limiting step in structure determination by X-ray diffraction. Even if single crystals are available, inadequate physical quality may seriously limit the resolution of the available data and consequently the accuracy of the atomic model. Recent studies show that targeted mutagenesis of surface patches containing residues with large flexible side chains and their...
Phosphotransacetylase (Pta) [EC 2.3.1.8] is ubiquitous in the carbon assimilation and energy-yielding pathways in anaerobic prokaryotes where it catalyzes the reversible transfer of the acetyl group from acetyl phosphate to CoA forming acetyl CoA and inorganic phosphate. The crystal structure of Pta from the methane-producing archaeon Methanosarcina thermophila, representing the first crystal structure...
A multifaceted approach is adopted to characterize EstA from Aspergillus niger (Bourne et al., 2004). Collectively, biophysical, bioinformatic, and biochemical analyses identify EstA as the lead member of a new class of fungal esterases within the superfamily of α/β-hydrolases.
The tRNA(Gm18) methyltransferase (TrmH) catalyzes the 2′-O methylation of guanosine 18 (Gua18) of tRNA. We solved the crystal structure of Thermus thermophilus TrmH complexed with S-adenosyl-L-methionine at 1.85 Å resolution. The catalytic domain contains a deep trefoil knot, which mutational analyses revealed to be crucial for the formation of the catalytic site and the cofactor binding pocket. The...
From the fungus Aspergillus niger, we identified a new gene encoding protein EstA, a member of the α/β-hydrolase fold superfamily but of unknown substrate specificity. EstA was overexpressed and its crystal structure was solved by molecular replacement using a lipase-acetylcholinesterase chimera template. The 2.1 Å resolution structure of EstA reveals a canonical Ser/Glu/His catalytic triad located...
High-throughput data collection for macromolecular crystallography requires an automated sample mounting and alignment system for cryo-protected crystals that functions reliably when integrated into protein-crystallography beamlines at synchrotrons. Rapid mounting and dismounting of the samples increases the efficiency of the crystal screening and data collection processes, where many crystals can...
The adapter protein ADAP (FYB/SLAP-130) provides a critical link between T cell receptor (TCR) signaling and cell adhesion via the activation of integrins. The C-terminal 70 residues of ADAP show homology to SH3 domains; however, conserved residues of the fold are absent. An alignment and annotation of this domain has therefore been elusive. We have solved the three-dimensional structure of the ADAP...
Copper-dependent enzymes are essential for many cellular processes, yet paradoxically, copper is highly cytotoxic. Bertini and coworkers (Arnesano et al., 2004) provide important new structural insight into the role of a copper chaperone in delivering effectively copper to the correct target.
In bacteriophage T4, the WXY system repairs DNA damage by a process that involves homologous recombination. This system comprises three proteins, the RecA-like recombination protein UvsX, a recombination mediator protein UvsY, and a helicase UvsW. Here we report the 2.0 Å resolution crystal structure of the N-terminal two domains of the UvsW helicase (UvsWNF; residues 1–282). The structure reveals...
Crystal structures of bacterial reaction center second-site revertants (Xu et al., 2004) show that proton transfer energetics and connectivity are both important when this membrane protein reforms a proton transfer pathway broken by site-directed mutation.
In the photosynthetic reaction center (RC) from Rhodobacter sphaeroides, the reduction of a bound quinone molecule Q B is coupled with proton uptake. When Asp-L213 is replaced by Asn, proton transfer is inhibited. Proton transfer was restored by two second-site revertant mutations, Arg-M233→Cys and Arg-H177→His. Kinetic effects of Cd 2+ on proton transfer showed that the entry point...
We report the solution structure of the Cro protein from bacteriophage P22. Comparisons of its sequence and structure to those of λ Cro strongly suggest an α-to-β secondary structure switching event during Cro evolution. The folds of P22 Cro and λ Cro share a three α helix fragment comprising the N-terminal half of the domain. However, P22 Cro's C terminus folds as two helices, while λ Cro's folds...
Erythroid spectrin, a major component of the cytoskeletal network of the red cell which contributes to both the stability and the elasticity of the red cell membrane, is composed of two subunits, α and β, each formed by 16–20 tandem repeats. The properties of the repeats and their relative arrangement are thought to be key determinants of spectrin flexibility. Here we report a 2.4 Å resolution crystal...
Recognition of and discrimination between potential glyco-substrates is central to the function of galectins. Here we dissect the fundamental parameters responsible for such selectivity by the fungal representative, CGL2. The 2.1 Å crystal structure of CGL2 and five substrate complexes reveal that this prototype galectin achieves increased substrate specificity by accommodating substituted oligosaccharides...
The crystal structure of the endonuclease, ribonuclease III, in complex with a double-stranded RNA fragment reveals the mechanism that allows for switching between two functional forms of the enzyme.
Arginine deiminase (ADI), an enzyme that hydrolyzes arginine to generate energy in many parasitic microorganisms, has potent anticancer activities and can halt growth of solid tumors. We determined the crystal structure of ADI from Mycoplasma arginini in two different forms (1.6 and 2.0 Å resolution) using multiple isomorphous replacement. ADI shares common structural features with the arginine-catabolizing...
Agarose is a gel-forming polysaccharide with an α-L(1,4)-3,6-anhydro-galactose, β-D(1,3)-galactose repeat unit, from the cell walls of marine red algae. β-agarase A, from the Gram-negative bacterium Zobellia galactanivorans, is secreted to the external medium and degrades agarose with an endo-mechanism. The structure of the inactive mutant β-agarase A-E147S in complex with agaro-octaose has been solved...
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