The three-dimensional structures support evidence that the Nef-Fyn complex forms in vivo and may have a crucial role in the T cell perturbating action of Nef by altering T cell receptor signaling. The structures of bound and unbound Nef reveal that the multivalency of SH3 binding may be achieved by a ligand induced flexibility in the RT loop. The structures suggest possible targets for the design of inhibitors which specifically block Nef-SH3 interactions.

Dozens of proteins capture, polymerize and reshape the clathrin lattice during clathrin-mediated endocytosis (CME). How or if this ensemble of proteins is organized in relation to the clathrin coat is unknown. Here, we map key molecules involved in CME at the nanoscale using correlative super-resolution light and transmission electron microscopy. We localize 19 different endocytic proteins (amphiphysin1, AP2, β2-arrestin, CALM, clathrin, DAB2, dynamin2, EPS15, epsin1, epsin2, FCHO2, HIP1R, intersectin, NECAP, SNX9, stonin2, syndapin2, transferrin receptor, VAMP2) on thousands of individual clathrin structures, generating a comprehensive molecular architecture of endocytosis with nanoscale precision. We discover that endocytic proteins distribute into distinct spatial zones in relation to the edge of the clathrin lattice. The presence or concentrations of proteins within these zones vary at distinct stages of organelle development. We propose that endocytosis is driven by the recruitment, reorganization and loss of proteins within these partitioned nanoscale zones.

Membrane fission is a fundamental process in the regulation and remodelling of cell membranes. Dynamin, a large GTPase, mediates membrane fission by assembling around, constricting and cleaving the necks of budding vesicles. Here we report a 3.75 Å resolution cryo-electron microscopy structure of the membrane-associated helical polymer of human dynamin-1 in the GMPPCP-bound state. The structure defines the helical symmetry of the dynamin polymer and the positions of its oligomeric interfaces, which were validated by cell-based endocytosis assays. Compared to the lipid-free tetramer form, membrane-associated dynamin binds to the lipid bilayer with its pleckstrin homology domain (PHD) and self-assembles across the helical rungs via its guanine nucleotide-binding (GTPase) domain. Notably, interaction with the membrane and helical assembly are accommodated by a severely bent bundle signalling element (BSE), which connects the GTPase domain to the rest of the protein. The BSE conformation is asymmetric across the inter-rung GTPase interface, and is unique compared to all known nucleotide-bound states of dynamin. The structure suggests that the BSE bends as a result of forces generated from the GTPase dimer interaction that are transferred across the stalk to the PHD and lipid membrane. Mutations that disrupted the BSE kink impaired endocytosis. We also report a 10.1 Å resolution cryo-electron microscopy map of a super-constricted dynamin polymer showing localized conformational changes at the BSE and GTPase domains, induced by GTP hydrolysis, that drive membrane constriction. Together, our results provide a structural basis for the mechanism of action of dynamin on the lipid membrane.

Understanding the issue of specificity imposed in the interactions of SH3 domains has largely been addressed in studies investigating the interaction of proline-rich amino acid sequences derived from potential ligands for these domains. Although the interaction with this motif forms an essential platform in the binding of SH3 domains, in many cases little specificity is observed and the difference in affinity for so-called specific and nonspecific proline-rich sequences is not great. Furthermore, the binding interface between an SH3 domain and a protein ligand appears to encompass more interactions than are represented by that involving the proline-rich motif. Here we investigate the issue of specificity from the opposite point of view; namely, how does a ligand recognize different SH3 domains? We present the crystal structure of the unbound SH3 domain from hemopoietic cell kinase (Hck) which is a member of the Src family of tyrosine kinases. This structure reveals that, unlike the structures of other Src kinase SH3 domains, the RT loop region is highly mobile and lacks a network of hydrogen bonds that is elsewhere apparent. The RT loop has been shown to form a major part of the binding interface between SH3 domains and HIV-1 Nef. Thermodynamic data, derived from isothermal titration calorimetry, for the binding of Hck SH3 to HIV-1 Nef show that the binding of Hck (KD = 1.5 microM) is approximately an order of magnitude tighter than those of other Src family kinases that were investigated (Fyn, Lck, and Src). This increase in affinity is attributed to, among other effects, the inherent flexibility in the RT loop which does not require breaking the network of hydrogen bonds to adopt the conformation required for binding.

HIV-1 replication requires Tsg101, a component of cellular endosomal sorting complex required for transport (ESCRT) machinery. Tsg101 possesses an ubiquitin (Ub) E2 variant (UEV) domain with a pocket that can bind PT/SAP motifs and another pocket that can bind Ub. The PTAP motif in the viral structural precursor polyprotein, Gag, allows the recruitment of Tsg101 and other ESCRTs to virus assembly sites where they mediate budding. It is not known how or even whether the UEV Ub binding function contributes to virus production. Here, we report that disruption of UEV Ub binding by commonly used drugs arrests assembly at an early step distinct from the late stage involving PTAP binding disruption. NMR reveals that the drugs form a covalent adduct near the Ub-binding pocket leading to the disruption of Ub, but not PTAP binding. We conclude that the Ub-binding pocket has a chaperone function involved in bud initiation.

A protein-encaged superparamagnetic iron oxide has been developed and characterized by using horse spleen apoferritin as a novel bioreactive environment. The roughly spherical magnetoferritin molecules, 120 A in diameter, are composed of a monocrystalline maghemite or magnetite core 73 A +/- 14 in diameter. Except for the additional presence of iron-rich molecules of higher molecular weight, the appearance and molecular weight (450 kd) of magnetoferritin are identical to that of natural ferritin; the molecules are externally indistinguishable from their precursor, with a pI (isoelectric point) in the range 4.3-4.6. The measured magnetic moment of the superparamagnetic cores is 13,200 Bohr magnetons per molecule, with T1 and T2 relaxivities (r1 and r2) of 8 and 175 L.mmol-1 (Fe).sec-1, respectively, at body temperature and clinical field strengths. The unusually high r2/r1 ratio of 22 is thought to arise from ideal core composition, with no evidence of crystalline paramagnetic inclusions. T2 relaxation enhancement can be well correlated to the field-dependent molecular magnetization, as given by the Langevin magnetization function, raised to a power in the range 1.4-1.6. With its nanodimensional biomimetic protein cage as a rigid, convenient matrix for complexing a plethora of bioactive substances, magnetoferritin may provide a novel template for specific targeting of selected cellular sites.

SacY is the prototype of a family of regulatory proteins able to prevent transcription termination. It interacts with a 29 nucleotide RNA sequence able to fold into a stem–loop structure and partially overlapping with a terminator sequence located in the 5′ leader mRNA region of the gene it controls. We show here that the N‐terminal fragment of SacY, SacY(1–55), and the corresponding fragments of other members of the family have antiterminator activities with efficiency and specificity identical to those of the full‐length proteins. In vitro, this activity correlates with the specific affinity of SacY(1–55) for its RNA target. UV melting experiments demonstrate that SacY(1–55) binding stabilizes the RNA target structure. The NMR solution structure of SacY(1–55) is very similar to that obtained in the crystal (van Tilbeurgh et al., 1997): the peptide is folded as a symmetrical dimer without any structural homology with other RNA‐binding domains yet characterized. According to a preliminary NMR analysis of the SacY(1–55)–RNA complex, the protein dimer is not disrupted upon RNA binding and several residues implicated in RNA recognition are located at the edge of the dimer interface. This suggests a new mode of protein–RNA interaction.

Unconventional myosin 15 is a molecular motor expressed in inner ear hair cells that transports protein cargos within developing mechanosensory stereocilia. Mutations of myosin 15 cause profound hearing loss in humans and mice; however, the properties of this motor and its regulation within the stereocilia organelle are unknown. To address these questions, we expressed a subfragment 1-like (S1) truncation of mouse myosin 15, comprising the predicted motor domain plus three light-chain binding sites. Following unsuccessful attempts to express functional myosin 15-S1 using the Spodoptera frugiperda (Sf9)-baculovirus system, we discovered that coexpression of the muscle-myosin-specific chaperone UNC45B, in addition to the chaperone heat-shock protein 90 (HSP90) significantly increased the yield of functional protein. Surprisingly, myosin 15-S1 did not bind calmodulin with high affinity. Instead, the IQ domains bound essential and regulatory light chains that are normally associated with class II myosins. We show that myosin 15-S1 is a barbed-end-directed motor that moves actin filaments in a gliding assay (∼430 nm·s −1 at 30°C), using a power stroke of 7.9 nm. The maximum ATPase rate (k cat ∼6 s ) was similar to the actin-detachment rate (k det = 6.2 s −1 ) determined in single molecule optical trapping experiments, indicating that myosin 15-S1 was rate limited by transit through strongly actin-bound states, similar to other processive myosin motors. Our data further indicate that in addition to folding muscle myosin, UNC45B facilitates maturation of an unconventional myosin. We speculate that chaperone coexpression may be a simple method to optimize the purification of other myosin motors from Sf9 insect cells.U nconventional myosin 15 is expressed by inner ear hair cells and accumulates at the tips of mechanosensory stereocilia (1, 2), actin-based organelles that are stimulated by sound and accelerations. A missense substitution in the myosin 15 motor domain results in abnormally short stereocilia in the Myo15 sh2/sh2