Xiao-Ping Xu

The structural basis of actin filament branching by the Arp2/3 complex.

The actin-related protein 2/3 (Arp2/3) complex mediates the formation of branched actin filaments at the leading edge of motile cells and in the comet tails moving certain intracellular pathogens. Crystal structures of the Arp2/3 complex are available, but the architecture of the junction formed by the Arp2/3 complex at the base of the branch was not known. In this study, we use electron tomography to reconstruct the branch junction with sufficient resolution to show how the Arp2/3 complex interacts with the mother filament. Our analysis reveals conformational changes in both the mother filament and Arp2/3 complex upon branch formation. The Arp2 and Arp3 subunits reorganize into a dimer, providing a short-pitch template for elongation of the daughter filament. Two subunits of the mother filament undergo conformational changes that increase stability of the branch. These data provide a rationale for why branch formation requires cooperative interactions among the Arp2/3 complex, nucleation-promoting factors, an actin monomer, and the mother filament.

Mechanism of Filament Nucleation and Branch Stability Revealed by the Structure of the Arp2/3 Complex at Actin Branch Junctions

Actin branch junctions are conserved cytoskeletal elements critical for the generation of protrusive force during actin polymerization-driven cellular motility. Assembly of actin branch junctions requires the Arp2/3 complex, upon activation, to initiate a new actin (daughter) filament branch from the side of an existing (mother) filament, leading to the formation of a dendritic actin network with the fast growing (barbed) ends facing the direction of movement. Using genetic labeling and electron microscopy, we have determined the structural organization of actin branch junctions assembled in vitro with 1-nm precision. We show here that the activators of the Arp2/3 complex, except cortactin, dissociate after branch formation. The Arp2/3 complex associates with the mother filament through a comprehensive network of interactions, with the long axis of the complex aligned nearly perpendicular to the mother filament. The actin-related proteins, Arp2 and Arp3, are positioned with their barbed ends facing the direction of daughter filament growth. This subunit map brings direct structural insights into the mechanism of assembly and mechanical stability of actin branch junctions.

Effects of Arp2 and Arp3 nucleotide-binding pocket mutations on Arp2/3 complex function

Contributions of actin-related proteins (Arp) 2 and 3 nucleotide state to Arp2/3 complex function were tested using nucleotide-binding pocket (NBP) mutants in Saccharomyces cerevisiae. ATP binding by Arp2 and Arp3 was required for full Arp2/3 complex nucleation activity in vitro. Analysis of actin dynamics and endocytosis in mutants demonstrated that nucleotide-bound Arp3 is particularly important for Arp2/3 complex function in vivo. Severity of endocytic defects did not correlate with effects on in vitro nucleation activity, suggesting that a critical Arp2/3 complex function during endocytosis may be structural rather than catalytic. A separate class of Arp2 and Arp3 NBP mutants suppressed phenotypes of mutants defective for actin nucleation. An Arp2 suppressor mutant increased Arp2/3 nucleation activity. Electron microscopy of Arp2/3 complex containing this Arp2 suppressor identified a structural change that also occurs upon Arp2/3 activation by nucleation promoting factors. These data demonstrate the importance of Arp2 and Arp3 nucleotide binding for nucleating activity, and Arp3 nucleotide binding for maintenance of cortical actin cytoskeleton cytoarchitecture.

Structural studies on full-length talin1 reveal a compact auto-inhibited dimer: implications for talin activation.

Talin is a large adaptor protein that activates integrins and couples them to cytoskeletal actin. Talin contains an N-terminal FERM (band 4.1, ezrin, radixin, moesin) domain (the head) linked to a flexible rod comprised of 13 amphipathic helical bundles (R1-R13) that terminate in a C-terminal helix (DD) that forms an anti-parallel dimer. We derived a three-dimensional structural model of full-length talin at a resolution of approximately 2.5nm using EM reconstruction of full-length talin and the known shapes of the individual domains and inter-domain angles as derived from small angle X-ray scattering. Talin adopts a compact conformation consistent with a dimer in which the two talin rods form a donut-shaped structure, with the two talin heads packed side by side occupying the hole at the center of this donut. In this configuration, the integrin binding site in the head domain and the actin-binding site at the carboxy-terminus of the rod are masked, implying that talin must unravel before it can support integrin activation and engage the actin cytoskeleton.

Three-dimensional reconstructions of Arp2/3 complex with bound nucleation promoting factors

Arp2/3 complex initiates the growth of branched actin‐filament networks by inducing actin polymerization from the sides of pre‐existing filaments. Nucleation promoting factors (NPFs) are essential for the branching reaction through interactions with the Arp2/3 complex prior to branch formation. The modes by which NPFs bind Arp2/3 complex and associated conformational changes have remained elusive. Here, we used electron microscopy to determine three‐dimensional structures at ∼2 nm resolution of Arp2/3 complex with three different bound NPFs: N‐WASp, Scar‐VCA and cortactin. All of these structures adopt a conformation with the two actin‐related proteins in an actin‐filament‐like dimer and the NPF bound to the pointed end. Distance constraints derived by fluorescence resonance energy transfer independently verified the NPF location. Furthermore, all bound NPFs partially occlude the actin‐filament binding site, suggesting that additional local structural rearrangements are required in the pathway of Arp2/3 complex activation to allow branch formation.

Holoenzyme structures of endothelial nitric oxide synthase - An allosteric role for calmodulin in pivoting the FMN domain for electron transfer

While the three-dimensional structures of heme- and flavin-binding domains of the NOS isoforms have been determined, the structures of the holoenzymes remained elusive. Application of electron cryo-microscopy and structural modeling of the bovine endothelial nitric oxide synthase (eNOS) holoenzyme produced detailed models of the intact holoenzyme in the presence and absence of Ca(2+)/calmodulin (CaM). These models accommodate the cross-electron transfer from the reductase in one monomer to the heme in the opposite monomer. The heme domain acts as the anchoring dimeric structure for the entire enzyme molecule, while the FMN domain is activated by CaM to move flexibly to bridge the distance between the reductase and oxygenase domains. Our results indicate that the key regulatory role of CaM involves the stabilization of structural intermediates and precise positioning of the pivot for the FMN domain tethered shuttling motion to accommodate efficient and rapid electron transfer in the homodimer of eNOS.

Efficient Extraction of Macromolecular Complexes from Electron Tomograms Based on Reduced Representation Templates

Electron tomography is the most widely applicable method for obtaining 3D information by electron microscopy. In the field of biology it has been realized that electron tomography is capable of providing a complete, molecular resolution three-dimensional mapping of entire proteoms. However, to realize this goal, information needs to be extracted efficiently from these tomograms. Owing to extremely low signal-to-noise ratios, this task is mostly carried out manually. Standard template matching approaches tend to generate large amounts of false positives. We developed an alternative method for feature extraction in biological electron tomography based on reduced representation templates, approximating the search model by a small number of anchor points used to calculate the scoring function. Using this approach we see a reduction of about 50% false positives with matched-filter approaches to below 5%. At the same time, false negatives stay below 5%, thus essentially matching the performance one would expect from human operators.

Orchestration of ErbB3 signaling through heterointeractions and homointeractions.

ErbB receptors form homodimers and heterodimers between family members. To model ErbB2/ErbB3 signaling, single-particle tracking data are used to create a simulation space with overlapping receptor domains. Stochastic modeling of receptor dimerization and phosphorylation reveals the complexity of ErbB2-3 interactions.

Validation methods for low-resolution fitting of atomic structures to electron microscopy data.

Fitting of atomic-resolution structures into reconstructions from electron cryo-microscopy is routinely used to understand the structure and function of macromolecular machines. Despite the fact that a plethora of fitting methods has been developed over recent years, standard protocols for quality assessment and validation of these fits have not been established. Here, we present the general concepts underlying current validation ideas as they relate to fitting of atomic-resolution models into electron cryo-microscopy reconstructions, with an emphasis on reconstructions with resolutions below the sub-nanometer range.

Probabilistic determination of probe locations from distance data.

Distance constraints, in principle, can be employed to determine information about the location of probes within a three-dimensional volume. Traditional methods for locating probes from distance constraints involve optimization of scoring functions that measure how well the probe location fits the distance data, exploring only a small subset of the scoring function landscape in the process. These methods are not guaranteed to find the global optimum and provide no means to relate the identified optimum to all other optima in scoring space. Here, we introduce a method for the location of probes from distance information that is based on probability calculus. This method allows exploration of the entire scoring space by directly combining probability functions representing the distance data and information about attachment sites. The approach is guaranteed to identify the global optimum and enables the derivation of confidence intervals for the probe location as well as statistical quantification of ambiguities. We apply the method to determine the location of a fluorescence probe using distances derived by FRET and show that the resulting location matches that independently derived by electron microscopy.