Type IV pili (Tfp) are widespread filamentous bacterial organelles that mediate multiple virulence-related phenotypes. They are composed mainly of pilin subunits, which are processed before filament assembly by dedicated prepilin peptidases. Other proteins processed by these peptidases, whose molecular nature and mode of action remain enigmatic, play critical roles in Tfp biology. We have performed a detailed structure/function analysis of one such protein, PilX from Neisseria meningitidis, which is crucial for formation of bacterial aggregates and adhesion to human cells. The x-ray crystal structure of PilX reveals the ␣/␤ roll fold shared by all pilins, and we show that this protein colocalizes with Tfp. These observations suggest that PilX is a minor, or low abundance, pilin that assembles within the filaments in a similar way to pilin. Deletion of a PilX distinctive structural element, which is predicted to be exposed on the filament surface, abolishes aggregation and adhesion. Our results support a model in which surface-exposed motifs in PilX subunits stabilize bacterial aggregates against the disruptive force of pilus retraction and illustrate how a minor pilus component can enhance the functional properties of pili of rather simple composition and structure.adhesion ͉ aggregation ͉ pilus retraction ͉ protein crystallography

Yellow fever mosquito sterol carrier protein (SCP-2) is known to bind to cholesterol. We report here the threedimensional structure of the complex of SCP-2 from Aedes aegypti with a C16 fatty acid to 1.35-Å resolution. The protein fold is exceedingly similar to the human and rabbit proteins, which consist of a five-stranded ␤-sheet that exhibits strand order 3-2-1-4-5 with an accompanying layer of four ␣-helices that cover the ␤-sheet. A large cavity exists at the interface of the layer ␣-helices and the ␤-sheet, which serves as the fatty acid binding site. The carboxylate moiety of the fatty acid is coordinated by a short loop that connects the first ␣-helix to the first ␤-strand, whereas the acyl chain extends deep into the interior of the protein. Interestingly, the orientation of the fatty acid is opposite to the observed orientation for Triton X-100 in the SCP-2-like domain from the peroxisomal multifunctional enzyme (Haapalainen, A. M., van

Site-directed mutagenesis and Laue diffraction data to 2.5 Å resolution were used to solve the structures of two sequential intermediates formed during the catalytic actions of isocitrate dehydrogenase. Both intermediates are distinct from the enzyme-substrate and enzyme-product complexes. Mutation of key catalytic residues changed the rate determining steps so that protein and substrate intermediates within the overall reaction pathway could be visualized.

Mosquito sterol carrier protein-2 (AeSCP-2) and sterol carrier protein-2-like2 (AeSCP-2L2) are members of the SCP-2 protein family with similar expression profiles in the mosquito life cycle. In an effort to understand how lipids can be transported by different SCP-2 proteins, the three-dimensional crystal structure of AeSCP-2L2 was solved at 1.7 Å resolution. AeSCP-2L2 forms a dimer and binds three fatty acids, one of which resides in a position within the internal cavity at a right angle to the others. This first report of ligand-bound dimerized protein in the SCP-2 protein family indicates that the family has a much more divergent mode of interaction with ligands than previously reported. The potential function of AeSCP-2L2 was investigated via in vivo incorporation of [ 3 H]cholesterol and [ 3 H]palmitic acid. Overexpression of AeSCP-2L2 in mosquito cells leads to an increased uptake of free fatty acid, whereas knockdown of AeSCP-2L2 in adult females decreases the accumulation of free fatty acid in the fat body from a blood meal. In contrast, overexpression or knockdown of AeSCP-2L2 has no effect on cholesterol uptake. Our results suggest that the main function of AeSCP-2L2 is as a general intracellular fatty acid carrier, as opposed to having a dedicated role in cholesterol transport.-Dyer, D. H., V. Wessely, K. T. Forest, and Q. Lan. Three-dimensional structure/function analysis of SCP-2-like2 reveals differences among SCP-2 family members. J. Lipid Res. 2008. 49: 644-653.

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