Dynamin is a mechanochemical GTPase that oligomerizes around the neck of clathrin-coated pits and catalyses vesicle scission in a GTP-hydrolysis-dependent manner. The molecular details of oligomerization and the mechanism of the mechanochemical coupling are currently unknown. Here we present the crystal structure of human dynamin 1 in the nucleotide-free state with a four-domain architecture comprising the GTPase domain, the bundle signalling element, the stalk and the pleckstrin homology domain. Dynamin 1 oligomerized in the crystals via the stalks, which assemble in a criss-cross fashion. The stalks further interact via conserved surfaces with the pleckstrin homology domain and the bundle signalling element of the neighbouring dynamin molecule. This intricate domain interaction rationalizes a number of disease-related mutations in dynamin 2 and suggests a structural model for the mechanochemical coupling that reconciles previous models of dynamin function.

SignificanceUnderstanding the formation and structure of protective bacterial biofilms will help to design and identify antimicrobial strategies. Our experiments with the secreted major biofilm protein TasA characterize on a molecular level in vivo the transition of a folded protein into protease-resistant biofilm-stabilizing fibrils. Such conformational changes from a globular state into fibrillar structures are so far not seen for other biofilm-forming proteins. In this context, TasA can serve as a model system to study functional fibril formation from a globular state.

Interaction mapping is a powerful strategy to elucidate the biological function of protein assemblies and their regulators. Here, we report the generation of a quantitative interaction network, directly linking 14 human proteins to the AAA+ ATPase p97, an essential hexameric protein with multiple cellular functions. We show that the high-affinity interacting protein ASPL efficiently promotes p97 hexamer disassembly, resulting in the formation of stable p97:ASPL heterotetramers. High-resolution structural and biochemical studies indicate that an extended UBX domain (eUBX) in ASPL is critical for p97 hexamer disassembly and facilitates the assembly of p97:ASPL heterotetramers. This spontaneous process is accompanied by a reorientation of the D2 ATPase domain in p97 and a loss of its activity. Finally, we demonstrate that overproduction of ASPL disrupts p97 hexamer function in ERAD and that engineered eUBX polypeptides can induce cell death, providing a rationale for developing anti-cancer polypeptide inhibitors that may target p97 activity.

Nck proteins are essential Src homology (SH) 2 and SH3 domain-bearing adapters that modulate actin cytoskeleton dynamics by linking proline-rich effector molecules to tyrosine kinases or phosphorylated signaling intermediates. Two mammalian pathogens, enteropathogenic Escherichia coli and vaccinia virus, exploit Nck as part of their infection strategy. Conflicting data indicate potential differences in the recognition specificities of the SH2 domains of the isoproteins Nck1 (Nck␣) and Nck2 (Nck␤ and Grb4). We have characterized the binding specificities of both SH2 domains and find them to be essentially indistinguishable. Crystal structures of both domains in complex with phosphopeptides derived from the enteropathogenic E. coli protein Tir concur in identifying highly conserved, specific recognition of the phosphopeptide. Differential peptide recognition can therefore not account for the preference of either Nck in particular signaling pathways. Binding studies using sequentially mutated, high affinity phosphopeptides establish the sequence variability tolerated in peptide recognition. Based on this binding motif, we identify potential new binding partners of Nck1 and Nck2 and confirm this experimentally for the Arf-GAP GIT1.Dynamic processes in eukaryotic cells, such as cellular movement, changes in cell shape, and transport of vesicles, rely on constant remodeling of the actin cytoskeleton. Adapter proteins, essential in transmitting and modulating corresponding stimuli, frequently contain SH2 3 domains to recognize and bind tyrosine-phosphorylated motifs. Nck1 (Nck␣) and Nck2 (Nck␤ or Grb4) are two such adapter proteins (1-3), both bearing three SH3 domains and a C-terminal SH2 domain (4). Mice lacking both Nck genes are not viable, underscoring the importance of these adapters (1). A high sequence identity (68% overall and 82% for the SH2 domains) and single gene knockouts of Nck1 and Nck2(1) indicate that the function of the proteins may substantially overlap. Both bind receptor tyrosine kinases such as the PDGFR (5) and other tyrosine-phosphorylated proteins via their SH2 domains (3). However, Nck1 or Nck2 has also been reported to bind distinct targets. Exclusive Nck2 binders include EphrinB1 (6, 7), EphrinB2 (8), and Disabled-1 (Dab-1) (9), all involved in neuronal signaling. In the case of the PDGFR, Tyr (P) 751 is reported to be Nck1-specific (5), whereas Tyr(P) 1009 isNck2-specific (10). Furthermore, Nck1 and Nck2 have both been implicated in the infection process of enteropathogenic Escherichia coli (EPEC) (11), a frequent cause of severe infant diarrhea (12). EPEC adheres tightly to the membrane of intestinal enterocytes inducing massive remodeling of the microfilament system and suppression of microvilli (13,14). This involves the "translocated intimin receptor" (Tir), introduced into the host cell by a type III secretion system (11). Insertion of Tir into the host cell membrane (15) provides a binding site to the bacterial outer membrane protein intimin (16). Tir clustering induces phosphoryla...

Summary Bacteriophages use specific tail proteins to recognize host cells. It is still not understood to molecular detail how the signal is transmitted over the tail to initiate infection. We have analysed in vitro DNA ejection in long‐tailed siphovirus 9NA and short‐tailed podovirus P22 upon incubation with Salmonella typhimurium lipopolysaccharide (LPS). We showed for the first time that LPS alone was sufficient to elicit DNA release from a siphovirus in vitro. Crystal structure analysis revealed that both phages use similar tailspike proteins for LPS recognition. Tailspike proteins hydrolyse LPS O antigen to position the phage on the cell surface. Thus we were able to compare in vitro DNA ejection processes from two phages with different morphologies with the same receptor under identical experimental conditions. Siphovirus 9NA ejected its DNA about 30 times faster than podovirus P22. DNA ejection is under control of the conformational opening of the particle and has a similar activation barrier in 9NA and P22. Our data suggest that tail morphology influences the efficiencies of particle opening given an identical initial receptor interaction event.