We report here 7 new mutations in the ADAMTS13 gene responsible for UpshawSchulman syndrome (USS), a catastrophic phenotype of congenital thrombotic thrombocytopenic purpura, by analyzing 5 Japanese families. There were 3 mutations that occurred at exon-intron boundaries: 414؉1G>A at intron 4, 686؉1G>A at intron 6, and 1244؉2T>G at intron 10 (numbered from the A of the initiation Met codon), and we confirmed that 2 of these mutations produced aberrantly spliced messenger RNAs (mRNAs). The remaining 4 mutations were missense mutations: R193W, I673F, C908Y, and R1123C. In expression experiments using HeLa cells, all mutants showed no or a marginal secretion of ADAMTS13. Taken together with the findings in our recent report we determined the responsible mutations in a total of 7 Japanese patients with USS with a uniform clinical picture of severe neonatal hyperbilirubinemia, and in their family members, based on ADAMTS13 gene analysis. Of these patients, 2 were homozygotes and 5 were compound heterozygotes. The parents of one homozygote were related (cousins), while those of the other were not. Molecular models of the metalloprotease, fifth domain of thrombospondin 1 (Tsp1-5), and Tsp1-8 domains of ADAMTS13 suggest that the missense mutations could cause structural defects in the mutants. IntroductionThrombotic thrombocytopenic purpura (TTP) is a life-threatening generalized disorder, and its diagnosis is made according to the criteria of Moschcowitz's pentad 1 : thrombocytopenia, microangiopathic hemolytic anemia (MAHA), fluctuating neurologic signs, renal failure, and fever. These criteria, however, are almost undistinguishable from those of hemolytic-uremic syndrome (HUS) with Gasser's triad 2 ; MAHA, thrombocytopenia, and renal insufficiency. Thus, the comprehensive term "TTP/HUS" or "thrombotic microangiopathy" 3 has frequently been used in clinical practice.Recent advances in elucidating the proteolytic processing of plasma von Willebrand factor (VWF) multimers have established assays for the activity of VWF-cleaving protease and its inhibitor (autoantibody). [4][5][6][7] These assays have largely made it possible to distinguish TTP from HUS, because the former has defective VWF-cleaving activity, whereas the latter has VWF-cleaving activity. 6,7 Studies by several groups of investigators have led to the identification of this enzyme as a new metalloprotease belonging to the ADAMTS (a disintegrinlike and metalloprotease with thrombospondin type 1 motif) family, which has been designated ADAMTS13. [8][9][10][11][12] This enzyme is produced in the liver. [10][11][12] The deduced amino acid residue number is 1427, and the gene contains 29 exons and is located on chromosome 9q34. [10][11][12] Upshaw-Schulman syndrome (USS) was originally reported as a disease complex with repeated episodes of thrombocytopenia and hemolytic anemia that quickly respond to infusions of fresh frozen plasma (FFP). [13][14][15][16] Clinical signs often develop in the patients during the newborn period or early infancy. In fact, the ea...

The CAPRI and CASP prediction experiments have demonstrated the power of community wide tests of methodology in assessing the current state of the art and spurring progress in the very challenging areas of protein docking and structure prediction. We sought to bring the power of community wide experiments to bear on a very challenging protein design problem that provides a complementary but equally fundamental test of current understanding of protein-binding thermodynamics. We have generated a number of designed protein-protein interfaces with very favorable computed binding energies but which do not appear to be formed in experiments, suggesting there may be important physical chemistry missing in the energy calculations. 28 research groups took up the challenge of determining what is missing: we provided structures of 87 designed complexes and 120 naturally occurring complexes and asked participants to identify energetic contributions and/or structural features that distinguish between the two sets. The community found that electrostatics and solvation terms partially distinguish the designs from the natural complexes, largely due to the non-polar character of the designed interactions. Beyond this polarity difference, the community found that the designed binding surfaces were on average structurally less embedded in the designed monomers, suggesting that backbone conformational rigidity at the designed surface is important for realization of the designed function. These results can be used to improve computational design strategies, but there is still much to be learned; for example, one designed complex, which does form in experiments, was classified by all metrics as a non-binder.

Community-wide blind prediction experiments such as CAPRI and CASP provide an objective measure of the current state of predictive methodology. Here we describe a community-wide assessment of methods to predict the effects of mutations on protein-protein interactions. Twenty-two groups predicted the effects of comprehensive saturation mutagenesis for two designed influenza hemagglutinin binders and the results were compared with experimental yeast display enrichment data obtained using deep sequencing. The most successful methods explicitly considered the effects of mutation on monomer stability in addition to binding affinity, carried out explicit side chain sampling and backbone relaxation, and evaluated packing, electrostatic and solvation effects, and correctly identified around a third of the beneficial mutations. Much room for improvement remains for even the best techniques, and large-scale fitness landscapes should continue to provide an excellent test bed for continued evaluation of methodological improvement.

We present the results for CAPRI Round 50, the fourth joint CASP-CAPRI protein assembly prediction challenge. The Round comprised a total of twelve targets, including six dimers, three trimers, and three higher-order oligomers. Four of these were easy targets, for which good structural templates were available either for the full assembly, or for the main interfaces (of the higher-order oligomers). Eight were

The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI-2) is essential for the intracellular survival and replication of Salmonella enterica. The expression of SPI-2 genes is dependent on a two-component regulatory system, SsrA (SpiR)/SsrB, encoded in the SPI-2 region. This paper shows that SlyA regulates transcription of the sensor kinase SsrA by binding to the ssrA promoter, indicating that SlyA is directly involved in the regulation of SPI-2 gene expression. A structure model of the SlyA dimer in complex with DNA was constructed. The model of SlyA indicated that its structure is very similar to that of other MarR family proteins. Based on this model, site-directed mutagenesis of residues located in the winged-helix region required for DNA binding and in the a-helices of the N-terminal and C-terminal regions required for dimerization of the SlyA protein was performed to identify the residues that are critical for SlyA function. Nine mutants of SlyA with single substitutions were unable to activate ssrA transcription in vivo. These mutant SlyA proteins revealed that the residues Leu-63, Val-64, Arg-65, Leu-67, Leu-70, Arg-86 and Lys-88 within the winged-helix region are required for DNA binding, and residues Leu-12 and Leu-126 within the a-helices of the N-terminal and C-terminal regions are required for efficient dimer formation. A Salmonella slyA mutant strain carrying a plasmid expressing SlyA derivatives containing mutations at these amino acid positions did not exhibit restored SlyA function in infected HeLa cells, thereby confirming the structural and functional relationships of the SlyA protein. INTRODUCTIONSalmonella enterica, a facultative intracellular pathogen, can replicate within macrophages. The intracellular survival of S. enterica serovar Typhimurium requires a number of virulence-associated proteins encoded in Salmonella pathogenicity island 2 (SPI-2) on the bacterial chromosome (Cirillo et al., 1998;Hensel, 2000;Ochman et al., 1996;Shea et al., 1996). SPI-2 encodes a two-component regulatory system, structural components of a type III secretion system (TTSS), effector proteins and chaperones, which mediate the secretion of the effectors (Cirillo et al., 1998;Hensel et al., 1998;Ochman et al., 1996;Shea et al., 1996). In infected macrophages, Salmonella replicates within a membranebound compartment (Salmonella-containing vacuole, SCV) inside the host cells (Steele-Mortimer et al., 2002). Intracellular activation of SPI-2 function is essential for SCV maturation along the phagolysosomal fusion pathway, which is responsible for the inhibition of bacterial degradation in phagolysosomes. The SPI-2 TTSS functions to export effector proteins across the SCV membrane and into the cytosol of host cells, resulting in the prevention of the recruitment of NADPH oxidase to the SCV (Gallois et al., 2001;Vazquez-Torres et al., 2000), as well as leading to the rearrangement of cellular microfilaments (Meresse et al., 2001;Miao et al., 2002) and microtubule networks (Brumell et al., 20...