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The plant cell wall degrading enzymes expressed by anaerobic microorganisms form large multienzyme complexes (cellulosomes). Cellulosomes assemble by the Type I dockerins on the catalytic subunits binding to the reiterated Type I cohesins in the molecular scaffold, while Type II dockerin-cohesin interactions anchor the complex onto the bacterial cell surface. Type I and Type II cohesin, dockerin pairs show no cross-specificity. Here we report the crystal structure of the Type II cohesin (CohII) from the Clostridium thermocellum cell surface anchoring protein SdbA. The protein domain contains nine beta-strands and a small alpha-helix. The beta-strands assemble into two elongated beta-sheets that display a typical jelly roll fold. The structure of CohII is very similar to Type I cohesins, and the dockerin binding site, which is centred at beta-strands 3, 5 and 6, is likely to be conserved in the two proteins. Subtle differences in the topology of the binding sites and a lack of sequence identity in the beta-strands that comprise the core of the dockerin binding site explain why Type I and Type II cohesins display such distinct specificities for their target dockerins.

We report the three-dimensional crystal structure of acidic seminal fluid protein (aSFP), a 12.9 kDa polypeptide of the spermadhesin family isolated from bovine seminal plasma, solved by the multiple isomorphous replacement method and refined with data to 1.9 A resolution with a final R-factor of 17.3%. aSFP is built by a single CUB domain architecture, a 100 to 110 amino-acid-residue extracellular module found in 16 functionally diverse proteins. The structure of aSFP reveals that the CUB domain displays a beta-sandwich topology organised into two 5-stranded beta-sheets, each of which contain two parallel and four antiparallel strands. The structure of aSFP is almost identical to that of porcine spermadhesins PSP-I and PSP-II, which in turn show limited structural similarity with jellyroll topologies of certain virus capsid proteins. Essentially, topologically conserved residues in these proteins are those internal amino acids forming the hydrophobic core of the CUB and the jellyroll domains, suggesting their importance in maintaining the integrity of these protein folds. On the other hand, the structure of aSFP shows structural features that are unique to this protein and which may provide a structural ground for understanding the distinct biological properties of different members of the spermadhesin protein family.

10 páginas, 4 figuras, 3 suplementos figuras -- PAGS nros. 571-580, The cellulosome is a highly elaborate cell-bound multienzyme complex that efficiently orchestrates the deconstruction of cellulose and hemicellulose, two of the nature's most abundant polymers. Understanding the intricacy of these nanomachines evolved by anaerobic microbes could sustain the development of an effective process for the conversion of lignocellulosic biomass to bio-ethanol. In Clostridium thermocellum, cellulosome assembly is mediated by high-affinity protein:protein interactions (> 109 M− 1) between dockerin modules found in the catalytic subunits and cohesin modules located in a non-catalytic protein scaffold termed CipA. Whereas the atomic structures of several cellulosomal components have been elucidated, the structural organization of the complete cellulosome remains elusive. Here, we reveal that a large fragment of the cellulosome presents a mostly compact conformation in solution, by solving the three-dimensional structure of a C. thermocellum mini-cellulosome comprising three consecutive cohesin modules, each bound to one Cel8A cellulase, at 35 Å resolution by cryo-electron microscopy. Interestingly, the three cellulosomal catalytic domains are found alternately projected outward from the CipA scaffold in opposite directions, in an arrangement that could expand the area of the substrate accessible to the catalytic domains. In addition, the cellulosome can transit from this compact conformation to a multitude of diverse and flexible structures, where the linkers between cohesin modules are extended and flexible. Thus, structural transitions controlled by changes in the degree of flexibility of linkers connecting consecutive cohesin modules could regulate the efficiency of substrate recognition and hydrolysis, This work was funded by the Spanish Ministry of Science and Innovation (SAF2008-00451 to O.L.), the Red Temática de Investigación Cooperativa en Cáncer from the Instituto de Salud Carlos III (RD06/0020/1001 to O.L.), the Autonomous Region of Madrid (CAM S-BIO-0214-2006 to O.L.), the Human Frontiers Science Program (RGP39/2008 to O.L.) and the Portuguese Science and Technology Foundation (project PTDC/QUI-BIQ/100359/2008). F.M.V.D. was supported by grant SFRH//BPD/26508/2006 from the Portuguese Science and Technology Foundation. B.G.A. has been supported by a JAE-DOC contract of the Consejo Superior de Investigaciones Científicas and a Juan de la Cierva contract from the Spanish Ministry of Science

Prostate-specific kallikrein, a member of the gene family of serine proteases, was initially discovered in semen and is the most useful serum marker for prostate cancer diagnosis and prognosis. We report the crystal structure at 1.42 A resolution of horse prostate kallikrein (HPK). This is the first structure of a serine protease purified from seminal plasma. HPK shares extensive sequence homology with human prostate-specific antigen (PSA), including a predicted chymotrypsin-like specificity, as suggested by the presence of a serine residue at position S1 of the specificity pocket. In contrast to other kallikreins, HPK shows a structurally distinct specificity pocket. Its entrance is blocked by the kallikrein loop, suggesting a possible protective or substrate-selective role for this loop. The HPK structure seems to be in an inactivated state and further processing might be required to allow the binding of substrate molecules. Crystal soaking experiments revealed a binding site for Zn2+ and Hg2+, two known PSA inhibitors.