SUMO proteins are ubiquitin-related modifiers implicated in the regulation of gene transcription, cell cycle, DNA repair, and protein localization. The molecular mechanisms by which the sumoylation of target proteins regulates diverse cellular functions remain poorly understood. Here we report isolation and characterization of SUMO1- and SUMO2-binding motifs. Using yeast two-hybrid system, bioinformatics, and NMR spectroscopy we define a common SUMO-interacting motif (SIM) and map its binding surfaces on SUMO1 and SUMO2. This motif forms a β-strand that could bind in parallel or antiparallel orientation to the β₂-strand of SUMO due to the environment of the hydrophobic core. A negative charge imposed by a stretch of neighboring acidic amino acids and/or phosphorylated serine residues determines its specificity in binding to distinct SUMO paralogues and can modulate the spatial orientation of SUMO-SIM interactions.

One of the principal peptide components of the amyloid plaque deposits of Alzheimer's disease in humans is the 40‐amino‐acid peptide β‐amyloid A4‐(1–40)‐peptide. The full‐length A4‐(1–40)‐peptide was chemically synthesized and the solution structure determined by two‐dimensional nuclear magnetic resonance spectroscopy and restrained molecular‐dynamics calculations. Synthetic human A4‐(1–40)‐peptide was soluble and non‐aggregating for several days in 40% (by vol.) trifluoroethanol/water. All spin systems could be unambiguously assigned, and a total of 203 sequential and medium‐range cross‐peaks were found in the NOESY (nuclear Overhauser enhancement spectroscopy) spectrum. Long‐range NOE cross‐peaks that would indicate tertiary structure of the peptide were absent. The main secondary‐structure elements found by chemical‐shift analysis, sequential and medium‐range NOESY data, and NOE‐based restrained molecular‐dynamics calculations were two helices, Gln15–Asp23 and IIe31–Met35, whereas the rest of the peptide was in random‐coil conformation. A similar secondary structure is suggested for the aggregation part of prions, the postulated causative agents of the transmissible spongiform encephalopathy. The sequence of the helical part of prion proteins was observed to be remarkably similar to the sequence of the helical part of human A4‐(1–40)‐peptide.

Wiskott Aldrich syndrome is a rare hereditary disease that affects cell morphology and signal transduction in hematopoietic cells. Different size fragments of the Wiskott Aldrich syndrome protein, W4, W7 and W13, were expressed in Escherichia coli or obtained from proteolysis. All contain the GTPase binding domain (GBD), also called Cdc42/Rac interactive binding region (CRIB), found in many putative downstream effectors of Rac and Cdc42. We have developed assays to measure the binding interaction between these fragments and Cdc42 employing fluorescentN-methylanthraniloyl-guanine nucleotide analogues. The fragments bind with submicromolar affinities in a GTP-dependent manner, with the largest fragment having the highest affinity, showing that the GBD/CRIB motif is necessary but not sufficient for tight binding. Rate constants for the interaction with W13 have been determined via surface plasmon resonance, and the equilibrium dissociation constant obtained from their ratio agrees with the value obtained by fluorescence measurements. Far UV circular dichroism spectra show significant secondary structure only for W13, supported by fluorescence studies using intrinsic protein fluorescence and quenching by acrylamide. Proton and ¹⁵N NMR measurements show that the GBD/CRIB motif has no apparent secondary structure and that the region C-terminal to the GBD/CRIB region is α-helical. The binding of Cdc42 induces a structural rearrangement of residues in the GBD/CRIB motif, or alternatively, the Wiskott Aldrich syndrome protein fragments have an ensemble of conformations, one of which is stabilized by Cdc42 binding. Thus, in contrast to Ras effectors, which have no conserved sequence elements but a defined domain structure with ubiquitin topology, Rac/Cdc42 effectors have a highly conserved binding region but no defined domain structure in the absence of the GTP-binding protein. Deviating from common belief GBD/CRIB is neither a structural domain nor sufficient for tight binding as regions outside this motif are necessary for structure formation and tight interaction with Rho/Rac proteins.

The processing of AVR3a from Phytophthora infestans shows striking similarities to the stepwise modification observed for the PEXEL effectors from the malaria p

The peptidyl-prolyl cis/trans isomerase hPin1 is a phosphorylation-dependent regulatory enzyme whose substrates are proteins involved in regulation of cell cycle, transcription, Alzheimer's disease, and cancer pathogenesis. We have determined the solution structure of the two domain protein hPin1-(1–163) and its separately expressed PPIase domain (50–163) (hPin1_PPIase) with an root mean square deviation of <0.5 Å over backbone atoms using NMR. Domain organization of hPin1 differs from that observed in structures solved by x-ray crystallography. Whereas PPIase and WW domain are tightly packed onto each other and share a common binding interface in crystals, our NMR-based data revealed only weak interaction of both domains at their interface in solution. Interaction between the two domains of full-length hPin1 is absent when the protein is dissected into the catalytic and the WW domain. It indicates that the flexible linker, connecting both domains, promotes binding. By evaluation of NOESY spectra we can show that the α1/β1 loop, which was proposed to undergo a large conformational rearrangement in the absence of sulfate and an Ala-Pro peptide, remained in the closed conformation under these conditions. Dissociation constants of 0.4 and 2.0 mm for sulfate and phosphate ions were measured at 12 °C by fluorescence spectroscopy. Binding of sulfate prevents hPin1 aggregation and changes surface charges across the active center and around the reactive and catalytically essential Cys¹¹³. In the absence of sulfate and/or reducing agent this residue seems to promote aggregation, as observed in hPin1 solutions in vitro.