The granulin/epithelin protein motif has an unusual structure consisting of a parallel stack of beta-hairpins stapled together by six disulphide bonds. The new structure also contains a folding subdomain shared by small toxins, protease inhibitors as well as the EGF-like protein modules.

The stereoselectivity of the reaction of furan (1) with maleic anhydride (2) and maleimide (3) was studied experimentally and theoretically. Although the two reactions are highly similar with regard to their preference for endo and exo steroisomers, notable differences were experimentally observed and explained on the basis of calculated reaction-free energies and transition-state barriers. The experimental values of rate constants (k(1+2endo) = (1.75 +/- 0.48) x 10(-5); mol(-1) l s(-1); k(1+2exo) = (3.10 +/- 0.55) x 10(-5); mol(-1) l s(-1); k(1+3endo) = (1.93 +/- 0.082) x 10(-5); mol(-1) l s(-1), k(1+3exo) = (1.38 +/- 0.055) x 10(-5); mol(-1) l s(-1) all at 300 K) and the observed reaction course clearly confirm that neither of these reactions are prototypical examples of Diels-Alder [4 + 2] cycloadditions, whose dominant preference is for endo isomers. However, only by comparing their energetics-calculated at the CCSD(T) level of theory-with the analogous reactions involving cyclopentadiene (8) as a diene can these observations be understood. The low thermodynamic stability of furan [4 + 2] adducts opens retro-Diels-Alder reaction channels and overrules the very small kinetic preference (calculated and measured here) of initial formation for endo stereoisomers. On a macroscopic scale "an irregular"-thermodynamically more stable-exo stereoisomer was consequently observed as a dominant species.

We determined the solution structure of myristoylated Mason-Pfizer monkey virus matrix protein by NMR spectroscopy. The myristoyl group is buried inside the protein and causes a slight reorientation of the helices. This reorientation leads to the creation of a binding site for phosphatidylinositols. The interaction between the matrix protein and phosphatidylinositols carrying C8 fatty acid chains was monitored by observation of concentration-dependent chemical shift changes of the affected amino acid residues, a saturation transfer difference experiment and changes in 31P chemical shifts. No differences in the binding mode or affinity were observed with differently phosphorylated phosphatidylinositols. The structure of the matrix protein–phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] complex was then calculated with HADDOCK software based on the intermolecular nuclear Overhauser enhancement contacts between the ligand and the matrix protein obtained from a 13C-filtered/13C-edited nuclear Overhauser enhancement spectroscopy experiment. PI(4,5)P2 binding was not strong enough for triggering of the myristoyl-switch. The structural changes of the myristoylated matrix protein were also found to result in a drop in the oligomerization capacity of the protein.

Despite extensive data demonstrating that immature retroviral particle assembly can take place either at the plasma membrane or at a distinct location within the cytoplasm, targeting of viral precursor proteins to either assembly site still remains poorly understood. Biochemical data presented here suggest that Tctex-1, a light chain of the molecular motor dynein, is involved in the intracellular targeting of Mason-Pfizer monkey virus (M-PMV) polyproteins to the cytoplasmic assembly site. Comparison of the three-dimensional structures of M-PMV wild-type matrix protein (wt MA) with a single amino acid mutant (R55F), which redirects assembly from a cytoplasmic site to the plasma membrane, revealed different mutual orientations of their C-and N-terminal domains. This conformational change buries a putative intracellular targeting motif located between both domains in the hydrophobic pocket of the MA molecule, thereby preventing the interaction with cellular transport mechanisms.capsid assembly ͉ dynein motor ͉ matrix protein structure ͉ retrovirus ͉ transport G ag polyproteins are major structural subunits of immature retroviral capsids and contain the determinants that mediate interactions with viral genomic RNA as well as particle assembly. The molecular mechanisms that control the accumulation of Gag molecules at the sites of assembly vary among retroviruses. Based on the assembly site, retroviruses have been shown to follow two major morphogenic pathways (1). While alpharetroviruses, gammaretroviruses, and lentiviruses (C-type retroviruses) assemble immature capsids at the inner side of the plasma membrane, the capsids of betaretroviruses (B/D-type) are formed in the cytoplasm. It has been shown that MasonPfizer monkey virus (M-PMV), which is the prototype of the D-type retroviruses, assembles at the pericentriolar region of an infected cell (2). Numerous studies have demonstrated that the matrix protein (MA), located at the N terminus of the Gag polyprotein, is responsible for targeting the polyprotein precursors to the site of assembly and for mediating transport of immature retroviral particles to the plasma membrane where budding occurs (3). A subtle difference in the regulation of the transport process has been suggested, as the results from several laboratories indicate that the destination of polyprotein precursors can be altered by mutations within MA. Amino acid substitutions in several domains of HIV-1 MA dramatically reduced the efficiency of particle production and redirected the majority of them to cytoplasmic vacuoles (4). Similarly, a substitution of basic for acidic residues in helix A of HIV-1 MA caused relocation of virus assembly to intracellular locations and produced normally budded noninfectious virions (5). Mutation of the N-terminal polybasic region of Moloney murine leukemia virus (Mo-MuLV) MA redirected virus assembly to the cytoplasm, suggesting a role of tryptophan residues in the intracellular transport (6).The N terminus of MA from most retroviruses, including M-PMV, is myristoylat...

The EGF-like domains in human thrombomodulin interact with and change the specificity of thrombin from a procoagulant enzyme to an anticoagulant enzyme. Recent experiments identified the minimal thrombin-binding region of thrombomodulin as the most acidic loop of the fifth EGF-like domain with a sequence of E408CPEGYILDDGFI420CTDIDE. High-resolution NMR spectroscopy was employed to characterize the interaction of a des-Ile420 thrombomodulin peptide, Cys1(409)Pro2Glu3Gly4Tyr5Ile6- Leu7Asp8Asp9Gly10Phe11Cys12Thr13Asp14Ile15Asp16Glu17(426), with its target coagulation protein, thrombin. The disulfide-bonded peptide was found to be structured only upon binding, while neither the linear nor the cyclized peptide exhibited any structural preference free in solution. The thrombin-bound structure of the cyclic thrombomodulin peptide was determined by transferred nuclear Overhauser effects (transferred NOEs) and by distance geometry and Monte Carlo calculations. The thrombin-bound cyclic peptide assumes an overall conformation similar to those observed in the free but intact EGF molecules. There is a type II beta-turn involving residues Pro2-Tyr5, followed by an optimized antiparallel beta-sheet involving residues Gly4-Asp8 and residues Phe11-Ile15. The thrombomodulin peptide provides a potential thrombin-binding surface between residues Tyr5 and Phe11, which are brought close by a chain reversal within the central beta-sheet. Comparison of the thrombin-bound structure of the EGF-like subdomain with other thrombin-peptide complexes revealed that a common thrombin-binding surface can be organized by different secondary structure elements with entirely different peptide sequences. The thrombin-bound structure of the thrombomodulin peptide may serve as a basis to understand the regulatory functions of thrombomodulin and as a guide for the design of specific inhibitors for thrombin.

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