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The Rho GDP-dissociation inhibitors (GDIs) negatively regulate Rho-family GTPases. The inhibitory activity of GDI derives both from an ability to bind the carboxy-terminal isoprene of Rho family members and extract them from membranes, and from inhibition of GTPase cycling between the GTP- and GDP-bound states. Here we demonstrate that these binding and inhibitory functions of rhoGDI can be attributed to two structurally distinct regions of the protein. A carboxy-terminal folded domain of relative molecular mass 16,000 (M[r] 16K) binds strongly to the Rho-family member Cdc42, yet has little effect on the rate of nucleotide dissociation from the GTPase. The solution structure of this domain shows a beta-sandwich motif with a narrow hydrophobic cleft that binds isoprenes, and an exposed surface that interacts with the protein portion of Cdc42. The amino-terminal region of rhoGDI is unstructured in the absence of target and contributes little to binding, but is necessary to inhibit nucleotide dissociation from Cdc42. These results lead to a model of rhoGDI function in which the carboxy-terminal binding domain targets the amino-terminal inhibitory region to GTPases, resulting in membrane extraction and inhibition of nucleotide cycling.

Cutinases are responsible for hydrolysis of the protective cutin lipid polyester matrix in plants and thus have been exploited for hydrolysis of small molecule esters and polyesters. Here we explore the reactivity, stability, and structure of Aspergillus oryzae cutinase and compare it to the well-studied enzyme from Fusarium solani. Two critical differences are highlighted in the crystallographic analysis of the A. oryzae structure: (i) an additional disulfide bond and (ii) a topologically favored catalytic triad with a continuous and deep groove. These structural features of A. oryzae cutinase are proposed to result in improved hydrolytic activity and altered substrate specificity profile, enhanced thermostability and remarkable reactivity towards the degradation of the synthetic polyester, polycaprolactone. The results presented here provide insight into engineering new cutinase-inspired biocatalysts with tailor-made properties.

We have used NMR spectroscopy to determine the solution structure of a complex between an oligonucleotide derived from stem IIB of the Rev responsive element (RRE-IIB) of HIV-1 mRNA and an in vivo selected, high affinity binding Arg-rich peptide. The peptide binds in a partially alpha-helical conformation into a pocket within the RNA deep groove. Comparison with the structure of a complex between an alpha-helical Rev peptide and RRE-IIB reveals that the sequence of the bound peptide determines the local conformation of the RRE peptide binding site. A conformational switch of an unpaired uridine base was revealed; this points out into the solvent in the Rev peptide complex, but it is stabilized inside the RNA deep groove by stacking with an Arg side chain in the selected peptide complex. The conformational switch has been visualized by NMR chemical shift mapping of the uridine H5/H6 atoms during a competition experiment in which Rev peptide was displaced from RRE-IIB by the higher affinity binding selected peptide.

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