Plasmodium vivax and Plasmodium knowlesi depend on the Duffy-Binding Protein DBL domain (RII-PvDBP or RII-PkDBP) engaging Duffy Antigen/Receptor for Chemokines on red blood cells during invasion. Inhibition of this key interaction provides an excellent opportunity for parasite control. There are competing models for whether Plasmodium ligands engage receptors as monomers or dimers, resolution of which has profound implications for parasite biology and control. We report crystallographic, solution and functional studies of RII-PvDBP, showing dimerization is required for and driven by receptor engagement. This work provides a unifying framework for prior studies and accounts for the action of naturally-acquired blocking-antibodies and the mechanism of immune evasion. We show dimerization is conserved in DBL-domain receptor-engagement, and propose receptor-mediated ligand-dimerization drives receptor affinity and specificity. Since dimerization is prevalent in signaling, our studies raise the possibility that induced dimerization activates pathways for invasion.

The human immunodeficiency virus type 1 (HIV-1) life cycle involves the reverse transcription of the viral RNA genome into cDNA and subsequent integration of the viral DNA by integrase (IN) into the host chromosomes. Although the amino acid sequences of INs differ significantly between viruses, INs share three conserved structural domains and their associated functions (13). The N-terminal domain (ϳ50 residues) contains a zinc-binding region (7), promotes multimerization (46), and is necessary for 3Ј-OH processing and strand transfer. The catalytic core domain (CCD) (ϳ162 residues) contains the highly conserved acidic D, D-35-E motif (27) that is involved in coordinating Mg 2ϩ for 3Ј-OH processing and strand transfer activities (4, 13). The catalytic core is also involved in target binding for strand transfer (3,20,26,40). The C-terminal domain (ϳ35 residues) binds to the viral DNA ϳ6 to 9 bp from the long terminal repeat (LTR) end (15); the C-terminus and CCD are also involved in IN multimerization (1, 24, 29a).IN, along with the reverse transcriptase and protease, is an antiretroviral target (2,35,38). Highly active antiretroviral therapy, consisting of various combinations of reverse transcriptase and protease inhibitors, has significantly decreased HIV-1 replication in humans. The emergence of multidrugresistant HIV-1 mutants and undesirable side effects associated with certain drug combinations necessitates continuing efforts to develop novel and effective combinational therapies. The addition of inhibitors of HIV-1 IN function would enhance highly active antiretroviral therapy. Raltegravir (MK-0518), an analog of the strand transfer inhibitor L-870,810 used in this report, is in phase III human clinical trials (18,33).Oligonucleotide-based assays in vitro have identified a large number of compounds that inhibit HIV-1 IN activities in vitro (25), the majority of which are ineffective at preventing HIV-1 replication in cell culture. The "strand transfer inhibitors" were identified as being effective against recombinant IN, suppressed HIV-1 replication in cell culture and in vivo, and were so named because of their selectivity in both cases towards inhibiting strand transfer over 16,21,22,38). The first generation of strand transfer inhibitors possessed a 1,3-diketo acid (DKA) pharmacophore, which served as a template in the development of the naphthyridine carboxamide inhibitors. They are structurally analogous to and function identically to DKA inhibitors but exhibit improved metabolic and pharmacokinetic properties and are represented here by compounds L-870,810 and L-870,812 (21, 23). DKA-mediated inhibition is accomplished by the contact of the DKA moiety with the divalent metal ion in the CCD of IN (19,38), and efficient inhibitor binding occurs only with IN bound to the viral DNA substrate (14,22,38

The translational diffusion constants, D, of trans-stilbene, 1,4-dipheny-1,3-butadiene, 1,6-dipheny-1,3,5-hexatriene, 1,1,4,4,-tetraphenyl-1,3-butadiene, tetraphenylethylene, 9,10-diphenylanthracene, p-terphenyl, bibenzyl, 1,1‘-binaphthyl, [2.2]paracyclophane, triptycene, and dodecahydrotriphenylene have been determined in the n-alkanes using capillary flow techniques. The solutes showed deviations from the Stokes−Einstein (SE) relation (D = k B T/(6πηr)); the values of the hydrodynamic radius, r, decrease as the viscosity, η, increases. The data can be fitted to D/T = A SE/η p with p < 1 (p = 1 for the SE relation). The values of p increase as the solute size increases; they range from p = 0.712 for p-terphenyl to p = 0.942 for 1,1,4,4,-tetraphenyl-1,3-butadiene. The deviations from SE behavior are discussed in terms of the ratio V s/V p, where V s and V p are the van der Waals volumes of a solvent and diffusing probe, respectively. The diffusion constants also are discussed in terms of the Wilke−Chang equation. The values of D -1 for several of the solutes are compared with their rotational correlation times, τθ, in the n-alkanes. The values of τθ, which showed deviations from the Stokes−Einstein−Debye expression (τθ = 4πr 3η/(3k B T)), have the same general dependence on viscosity as D -1.

Summary Bacterial pathogens use secreted effector proteins to subvert host-cell defenses. VopL is an effector protein from Vibrio parahaemolyticus that nucleates actin filaments. VopL consists of a VopL C-terminal Domain (VCD) and a tandem array of three WASP homology 2 (WH2) motifs. Here we report the crystal structure of the VCD dimer bound to actin. The VCD binds three actin monomers in a spatial arrangement close to that in the canonical actin filament. In this configuration each actin can readily accommodate a WH2 motif. The data suggest a mechanism of nucleation wherein VopL creates filament-like structures, organized by the VCD and delivered by the WH2 array, that can template addition of new monomers. Similarities with Arp2/3 complex and formin proteins suggest that organization of monomers into filament-like structures is a general and central feature of actin nucleation.

13C Methyl TROSY NMR spectroscopy has emerged as a powerful method for studying the dynamics of large systems such as macromolecular assemblies and membrane proteins. Specific 13C labeling of aliphatic methyl groups and perdeuteration has been limited primarily to proteins expressed in E. coli, preventing studies of many eukaryotic proteins of physiological and biomedical significance. We demonstrate the feasibility of efficient 13C isoleucine δ1-methyl labeling in a deuterated background in an established eukaryotic expression host, Pichia pastoris, and show that this method can be used to label the eukaryotic protein actin, which cannot be expressed in bacteria. This approach will enable NMR studies of previously intractable targets.

The "strand transfer inhibitors" of human immunodeficiency virus type-1 (HIV-1) integrase (IN), so named because of their pronounced selectivity for inhibiting strand transfer over 3 OH processing, block virus replication in vivo and ex vivo and prevent concerted integration in vitro. We explored the kinetics of product formation and strand transfer inhibition within reconstituted synaptic complexes capable of concerted integration. Synaptic complexes were formed with viral DNA donors containing either two blunt ends, two 3-OH-processed ends, or one of each. We determined that one blunt end within a synaptic complex is a sufficient condition for low-nanomolarrange strand

Chromosome segregation during cell division requires engagement of kinetochores of sister chromatids with microtubules emanating from opposite poles. As the corresponding microtubules shorten, these 'bioriented' sister kinetochores experience tension-dependent stabilization of microtubule attachments. The yeast XMAP215 family member and microtubule polymerase, Stu2, associates with kinetochores and contributes to tension-dependent stabilization in vitro. We show here that a C-terminal segment of Stu2 binds the four-way junction of the Ndc80 complex (Ndc80c) and that residues conserved both in yeast Stu2 orthologs and in their metazoan counterparts make specific contacts with Ndc80 and Spc24. Mutations that perturb this interaction prevent association of Stu2 with kinetochores, impair cell viability, produce biorientation defects, and delay cell cycle progression. Ectopic tethering of the mutant Stu2 species to the Ndc80c junction restores wild-type function in vivo. These findings show that the role of Stu2 in tension-sensing depends on its association with kinetochores by binding with Ndc80c.

The translational diffusion constants, D, of biphenyl, trans-stilbene, 1,4-diphenyl-1,3-butadiene, 1,1,4,4-tetraphenyl-1,3-butadiene, 1,6-diphenyl-1,3,5-hexatriene, tetraphenylethylene, 9,10-diphenylanthracene, bibenzyl, triptycene, perylene and 2,3-benzanthracene (tetracene) have been measured in combinations of the cycloalkanes cyclohexane, methylcyclohexane, nbutylcyclohexane, cis-decalin and trans-decalin using capillary flow techniques. Tetracene and chrycene have been studied in a series of n-alkanes. Deviations from the Stokes-Einstein (SE) relation (D ¼ k B T/6pr) were found. For a given solute, the hydrodynamic radius r decreases as both the viscosity and the solvent/solute size ratio increase; the data were fitted to D/ T ¼ A/ p with p<1 ( p ¼ 1 for the SE relation). The p values in the cycloalkanes increase as the solute size increases, are compared to the values in the n-alkanes and are discussed in terms of the properties of the two types of solvent. The experimental D values also are compared to the predictions of the Wilke-Chang equation and a free volume model which includes both the masses and sizes of the solution components.