LACTB is a filament-forming protein localized in mitochondria.

LACTB is a mammalian active-site serine protein that has evolved from a bacterial penicillin-binding protein. Penicillin-binding proteins are involved in the metabolism of peptidoglycan, the major bacterial cell wall constituent, implying that LACTB has been endowed with novel biochemical properties during eukaryote evolution. Here we demonstrate that LACTB is localized in the mitochondrial intermembrane space, where it is polymerized into stable filaments with a length extending more than a hundred nanometers. We infer that LACTB, through polymerization, promotes intramitochondrial membrane organization and micro-compartmentalization. These findings have implications for our understanding of mitochondrial evolution and function.

The structure of Neurospora crassa 3-carboxy-cis,cis-muconate lactonizing enzyme, a beta propeller cycloisomerase.

Muconate lactonizing enzymes (MLEs) convert cis,cis-muconates to muconolactones in microbes as part of the beta-ketoadipate pathway; some also dehalogenate muconate derivatives of xenobiotic haloaromatics. There are three different MLE classes unrelated by evolution. We present the X-ray structure of a eukaryotic MLE, Neurospora crassa 3-carboxy-cis,cis-muconate lactonizing enzyme (NcCMLE) at 2.5 A resolution, with a seven-bladed beta propeller fold. It is related neither to bacterial MLEs nor to other beta propeller enzymes, but is structurally similar to the G protein beta subunit. It reveals a novel metal-independent cycloisomerase motif unlike the bacterial metal cofactor MLEs. Together, the bacterial MLEs and NcCMLE structures comprise a striking structural example of functional convergence in enzymes for 1,2-addition-elimination of carboxylic acids. NcCMLE and bacterial MLEs may enhance the reaction rate differently: the former by electrophilic catalysis and the latter by electrostatic stabilization of the enolate.

The structural basis of receptor-binding by Escherichia coli associated with diarrhea and septicemia.

GafD in Escherichia coli G (F17) fimbriae is associated with diarrheal disease, and the structure of the ligand-binding domain, GafD1-178, has been determined at 1.7A resolution in the presence of the receptor sugar N-acetyl-D-glucosamine. The overall fold is a beta-barrel jelly-roll fold. The ligand-binding site was identified and localized to the side of the molecule. Receptor binding is mediated by side-chain as well main-chain interactions. Ala43-Asn44, Ser116-Thr117 form the sugar acetamide specificity pocket, while Asp88 confers tight binding and Trp109 appears to position the ligand. There is a disulfide bond that rigidifies the acetamide specificity pocket. The three fimbrial lectins, GafD, FimH and PapG share similar beta-barrel folds but display different ligand-binding regions and disulfide-bond patterns. We suggest an evolutionary path for the evolution of the very diverse fimbrial lectins from a common ancestral fold.

The structure of the bacteriophage PRD1 spike sheds light on the evolution of viral capsid architecture.

Comparisons of bacteriophage PRD1 and adenovirus protein structures and virion architectures have been instrumental in unraveling an evolutionary relationship and have led to a proposal of a phylogeny-based virus classification. The structure of the PRD1 spike protein P5 provides further insight into the evolution of viral proteins. The crystallized P5 fragment comprises two structural domains: a globular knob and a fibrous shaft. The head folds into a ten-stranded jelly roll beta barrel, which is structurally related to the tumor necrosis factor (TNF) and the PRD1 coat protein domains. The shaft domain is a structural counterpart to the adenovirus spike shaft. The structural relationships between PRD1, TNF, and adenovirus proteins suggest that the vertex proteins may have originated from an ancestral TNF-like jelly roll coat protein via a combination of gene duplication and deletion.

The Yersinia adhesin YadA collagen-binding domain structure is a novel left-handed parallel beta-roll.

The crystal structure of the recombinant collagen-binding domain of Yersinia adhesin YadA from Yersinia enterocolitica serotype O:3 was solved at 1.55 A resolution. The trimeric structure is composed of head and neck regions, and the collagen binding head region is a novel nine-coiled left-handed parallel beta-roll. Before the beta-roll, the polypeptide loops from one monomer to the rest, and after the beta-roll the neck region does the same, making the transition from the globular head region to the narrower stalk domain. This creates an intrinsically stable 'lock nut' structure. The trimeric form of YadA is required for collagen binding, and mutagenesis of its surface residues allowed identification of a putative collagen-binding surface. Furthermore, a new structure-sequence motif for YadA beta-roll was used to identify putative YadA-head-like domains in a variety of human and plant pathogens. Such domains may therefore be a common bacterial strategy for avoiding host response.

3-Carboxy-cis,cis-muconate lactonizing enzyme from Neurospora crassa: MAD phasing with 80 selenomethionines.

The structure of 3-carboxy-cis,cis-muconate lactonizing enzyme from Neurospora crassa was determined at 3.0 A resolution. Phase information was derived from a multiwavelength anomalous dispersion (MAD) experiment conducted at three wavelengths using crystals of fully substituted selenomethionine protein. However, the structure determination was not routine owing to the relatively poor quality of the diffraction data and the large number of twofolds in the unit cell. Eventually, 80 selenium sites were identified by the combined use of direct methods and real-space map interpretation. This represents one of the largest selenium substructures solved and used for phasing. Some of the difficulties in the structure determination and the methods used to address them are discussed.

Structural conservation between the actin monomer-binding sites of twinfilin and actin-depolymerizing factor (ADF)/cofilin.

Twinfilin is an evolutionarily conserved actin monomer-binding protein that regulates cytoskeletal dynamics in organisms from yeast to mammals. It is composed of two actin-depolymerization factor homology (ADF-H) domains that show approximately 20% sequence identity to ADF/cofilin proteins. In contrast to ADF/cofilins, which bind both G-actin and F-actin and promote filament depolymerization, twinfilin interacts only with G-actin. To elucidate the molecular mechanisms of twinfilin-actin monomer interaction, we determined the crystal structure of the N-terminal ADF-H domain of twinfilin and mapped its actin-binding site by site-directed mutagenesis. This domain has similar overall structure to ADF/cofilins, and the regions important for actin monomer binding in ADF/cofilins are especially well conserved in twinfilin. Mutagenesis studies show that the N-terminal ADF-H domain of twinfilin and ADF/cofilins also interact with actin monomers through similar interfaces, although the binding surface is slightly extended in twinfilin. In contrast, the regions important for actin-filament interactions in ADF/cofilins are structurally different in twinfilin. This explains the differences in actin-interactions (monomer versus filament binding) between twinfilin and ADF/cofilins. Taken together, our data show that the ADF-H domain is a structurally conserved actin-binding motif and that relatively small structural differences at the actin interfaces of this domain are responsible for the functional variation between the different classes of ADF-H domain proteins.

Crystallization and preliminary X-ray diffraction analysis of bacteriophage varphi12 packaging factor P7.

Bacteriophage varphi12 protein P7 is a structural component of the polymerase complex and ensures stable packaging of the genomic RNA. varphi12 P7 has been cloned, purified and crystallized. Crystals belong to space group P3(2)21, with unit-cell parameters a = 75.7, b = 75.7, c = 45.2 A, alpha = 90, beta = 90, gamma = 120 degrees , and diffract beyond 2.0 A. Multiple anomalous dispersion data have been collected from crystals of selenomethionylated P7. Mass spectroscopy showed proteolysis of the crystallized protein and a truncated form, P7DeltaC, gave crystals of similar morphology. Cross-linking experiments implicated the N-terminal domain of P7 as being essential for dimerization.