Structural basis for membrane recruitment and allosteric activation of cytohesin family Arf GTPase exchange factors.

Membrane recruitment of cytohesin family Arf guanine nucleotide exchange factors depends on interactions with phosphoinositides and active Arf GTPases that, in turn, relieve autoinhibition of the catalytic Sec7 domain through an unknown structural mechanism. Here, we show that Arf6-GTP relieves autoinhibition by binding to an allosteric site that includes the autoinhibitory elements in addition to the PH domain. The crystal structure of a cytohesin-3 construct encompassing the allosteric site in complex with the head group of phosphatidyl inositol 3,4,5-trisphosphate and N-terminally truncated Arf6-GTP reveals a large conformational rearrangement, whereby autoinhibition can be relieved by competitive sequestration of the autoinhibitory elements in grooves at the Arf6/PH domain interface. Disposition of the known membrane targeting determinants on a common surface is compatible with multivalent membrane docking and subsequent activation of Arf substrates, suggesting a plausible model through which membrane recruitment and allosteric activation could be structurally integrated.

Specificity and membrane partitioning of Grsp1 signaling complexes with Grp1 family Arf exchange factors.

The Arf exchange factor Grp1 selectively binds phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P(3)], which is required for recruitment to the plasma membrane in stimulated cells. The mechanisms for phosphoinositide recognition by the PH domain, catalysis of nucleotide exchange by the Sec7 domain, and autoinhibition by elements proximal to the PH domain are well-characterized. The N-terminal heptad repeats in Grp1 have also been shown to mediate homodimerization in vitro as well as heteromeric interactions with heptad repeats in the FERM domain-containing protein Grsp1 both in vitro and in cells [Klarlund, J. K., et al. (2001) J. Biol. Chem. 276, 40065-40070]. Here, we have characterized the oligomeric state of Grsp1 and Grp1 family proteins (Grp1, ARNO, and Cytohesin-1) as well as the oligomeric state, stoichiometry, and specificity of Grsp1 complexes with Grp1, ARNO, and Cytohesin-1. At low micromolar concentrations, Grp1 and ARNO are homodimeric whereas Cytohesin-1 and Grsp1 are monomeric. When mixed with Grsp1, Grp1 homodimers and Cytohesin-1 monomers spontaneously re-equilibrate to form heterodimers, whereas approximately 50% of ARNO remains homodimeric under the same conditions. Fluorescence resonance energy transfer experiments suggest that the Grsp1 heterodimers with Grp1 and Cytohesin-1 adopt a largely antiparallel orientation. Finally, formation of Grsp1-Grp1 heterodimers does not substantially influence the binding of Grp1 to the headgroups of PtdIns(3,4,5)P(3) or PtdIns(4,5)P(2), nor does it influence partitioning with liposomes containing PtdIns(3,4,5)P(3), PtdIns(4,5)P(2), and/or phosphatidylserine.

Structural Organization and Dynamics of Homodimeric Cytohesin Family Arf GTPase Exchange Factors in Solution and on Membranes.

Membrane dynamic processes require Arf GTPase activation by guanine nucleotide exchange factors (GEFs) with a Sec7 domain. Cytohesin family Arf GEFs function in signaling and cell migration through Arf GTPase activation on the plasma membrane and endosomes. In this study, the structural organization of two cytohesins (Grp1 and ARNO) was investigated in solution by size exclusion-small angle X-ray scattering and negative stain-electron microscopy and on membranes by dynamic light scattering, hydrogen-deuterium exchange-mass spectrometry and guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange assays. The results suggest that cytohesins form elongated dimers with a central coiled coil and membrane-binding pleckstrin-homology (PH) domains at opposite ends. The dimers display significant conformational heterogeneity, with a preference for compact to intermediate conformations. Phosphoinositide-dependent membrane recruitment is mediated by one PH domain at a time and alters the conformational dynamics to prime allosteric activation by Arf-GTP. A structural model for membrane targeting and allosteric activation of full-length cytohesin dimers is discussed.

Structural Dynamics Control Allosteric Activation of Cytohesin Family Arf GTPase Exchange Factors.

Membrane dynamic processes including vesicle biogenesis depend on Arf guanosine triphosphatase (GTPase) activation by guanine nucleotide exchange factors (GEFs) containing a catalytic Sec7 domain and a membrane-targeting module such as a pleckstrin homology (PH) domain. The catalytic output of cytohesin family Arf GEFs is controlled by autoinhibitory interactions that impede accessibility of the exchange site in the Sec7 domain. These restraints can be relieved through activator Arf-GTP binding to an allosteric site comprising the PH domain and proximal autoinhibitory elements (Sec7-PH linker and C-terminal helix). Small-angle X-ray scattering and negative-stain electron microscopy were used to investigate the structural organization and conformational dynamics of cytohesin-3 (Grp1) in autoinhibited and active states. The results support a model in which hinge dynamics in the autoinhibited state expose the activator site for Arf-GTP binding, while subsequent C-terminal helix unlatching and repositioning unleash conformational entropy in the Sec7-PH linker to drive exposure of the exchange site.

Expression and Structural Analyses of Human DNA Polymerase θ (POLQ).

DNA polymerase theta (pol θ) is an evolutionarily conserved protein encoded by the POLQ gene in mammalian genomes. Pol θ is the defining enzyme for a pathway of DSB repair termed "alternative end-joining" (altEJ) or "theta-mediated end-joining." This pathway contributes significantly to the radiation resistance of mammalian cells. It also modulates accuracy in repair of breaks that occur at stalled DNA replication forks, during diversification steps of the mammalian immune system, during repair of CRISPR-Cas9, and in many DNA integration events. Pol θ is a potentially important clinical target, particularly for cancers deficient in other break repair strategies. The enzyme is uniquely able to mediate joining of single-stranded 3' ends. Because of these unusual biochemical properties and its therapeutic importance, it is essential to study structures of pol θ bound to DNA. However, challenges for expression and purification are presented by the large size of pol θ (2590 residues in humans) and unusual juxtaposition of domains (a helicase-like domain and distinct DNA polymerase, separated by a region predicted to be largely disordered). Here we summarize work on the expression and purification of the full-length protein, and then focus on the design, expression, and purification of an active C-terminal polymerase fragment. The generation of this active construct was nontrivial and time consuming. Almost all published biochemical work to date has been performed with this domain fragment. Strategies to obtain and improve crystals of a ternary pol θ complex (enzyme:DNA:nucleotide) are also presented, along with key elements of the structure.

Methods for analysis of size-exclusion chromatography-small-angle X-ray scattering and reconstruction of protein scattering.

Size-exclusion chromatography in line with small-angle X-ray scattering (SEC-SAXS) has emerged as an important method for investigation of heterogeneous and self-associating systems, but presents specific challenges for data processing including buffer subtraction and analysis of overlapping peaks. This paper presents novel methods based on singular value decomposition (SVD) and Guinier-optimized linear combination (LC) to facilitate analysis of SEC-SAXS data sets and high-quality reconstruction of protein scattering directly from peak regions. It is shown that Guinier-optimized buffer subtraction can reduce common subtraction artifacts and that Guinier-optimized linear combination of significant SVD basis components improves signal-to-noise and allows reconstruction of protein scattering, even in the absence of matching buffer regions. In test cases with conventional SAXS data sets for cytochrome c and SEC-SAXS data sets for the small GTPase Arf6 and the Arf GTPase exchange factors Grp1 and cytohesin-1, SVD-LC consistently provided higher quality reconstruction of protein scattering than either direct or Guinier-optimized buffer subtraction. These methods have been implemented in the context of a Python-extensible Mac OS X application known as Data Evaluation and Likelihood Analysis (DELA), which provides convenient tools for data-set selection, beam intensity normalization, SVD, and other relevant processing and analytical procedures, as well as automated Python scripts for common SAXS analyses and Guinier-optimized reconstruction of protein scattering.

Grp1 plays a key role in linking insulin signaling to glut4 recycling.

The glucose transporter type 4 (glut4) is critical for metabolic homeostasis. Insulin regulates glut4 by modulating its expression on the cell surface. This regulation is mainly achieved by targeting the endocytic recycling of glut4. We identify general receptor for 3-phosphoinositides 1 (Grp1) as a guanine nucleotide exchange factor for ADP-ribosylation factor 6 (ARF6) that promotes glut4 vesicle formation. Grp1 also promotes the later steps of glut4 recycling through ARF6. Insulin signaling regulates Grp1 through phosphorylation by Akt. We also find that mutations that mimic constitutive phosphorylation of Grp1 can bypass upstream insulin signaling to induce glut4 recycling. Thus, we have uncovered a major mechanism by which insulin regulates glut4 recycling. Our findings also reveal the complexity by which a single small GTPase in vesicular transport can coordinate its multiple steps to accomplish a round of transport.