Julie Ménétrey

A Structural State of the Myosin V Motor without Bound Nucleotide

The myosin superfamily of molecular motors use ATP hydrolysis and actin-activated product release to produce directed movement and force. Although this is generally thought to involve movement of a mechanical lever arm attached to a motor core, the structural details of the rearrangement in myosin that drive the lever arm motion on actin attachment are unknown. Motivated by kinetic evidence that the processive unconventional myosin, myosin V, populates a unique state in the absence of nucleotide and actin, we obtained a 2.0 Å structure of a myosin V fragment. Here we reveal a conformation of myosin without bound nucleotide. The nucleotide-binding site has adopted new conformations of the nucleotide-binding elements that reduce the affinity for the nucleotide. The major cleft in the molecule has closed, and the lever arm has assumed a position consistent with that in an actomyosin rigor complex. These changes have been accomplished by relative movements of the subdomains of the molecule, and reveal elements of the structural communication between the actin-binding interface and nucleotide-binding site of myosin that underlie the mechanism of chemo-mechanical transduction.

The structure of the myosin VI motor reveals the mechanism of directionality reversal

Here we solve a 2.4-Å structure of a truncated version of the reverse-direction myosin motor, myosin VI, that contains the motor domain and binding sites for two calmodulin molecules. The structure reveals only minor differences in the motor domain from that in plus-end directed myosins, with the exception of two unique inserts. The first is near the nucleotide-binding pocket and alters the rates of nucleotide association and dissociation. The second unique insert forms an integral part of the myosin VI converter domain along with a calmodulin bound to a novel target motif within the insert. This serves to redirect the effective ‘lever arm’ of myosin VI, which includes a second calmodulin bound to an ‘IQ motif’, towards the pointed (minus) end of the actin filament. This repositioning largely accounts for the reverse directionality of this class of myosin motors. We propose a model incorporating a kinesin-like uncoupling/docking mechanism to provide a full explanation of the movements of myosin VI.

Structure of the Sec7 domain of the Arf exchange factor ARNO.

Small G proteins switch from a resting, GDP-bound state to an active, GTP-bound state. As spontaneous GDP release is slow, guanine-nucleotide-exchange factors (GEFs) are required to promote fast activation of small G proteins through replacement of GDP with GTP in vivo. Families of GEFs with no sequence similarity to other GEF families have now been assigned to most families of small G proteins. In the case of the small G protein Arf1, the exchange of bound GDP for GTP promotes the coating of secretory vesicles in Golgi traffic. An exchange factor for human Arf1, ARNO, and two closely related proteins, named cytohesin 1 (ref. 4) and GPS1 (ref. 5), have been identified. These three proteins are modular proteins with an amino-terminal coiled-coil, a central Sec7-like domain and a carboxy-terminal pleckstrin homology domain. The Sec7 domain contains the exchange-factor activity. It was first found in Sec7, a yeast protein involved in secretion, and is present in several other proteins, including the yeast exchange factors for Arf, Gea1 and Gea2 (refs 7–9). Here we report the crystal structure of the Sec7 domain of human ARNO at 2 Å resolution and the identification of the site of interaction of ARNO with Arf.

The structural GDP/GTP cycle of human Arf6

The small GTP‐binding protein Arf6 coordinates membrane traffic at the plasma membrane with aspects of cytoskeleton organization. This function does not overlap with that of other members of the ADP‐ribosylation factor (Arf) family, although their switch regions, which are their major sites of interaction with regulators and effectors, have virtually identical sequences. Here we report the crystal structure of full‐length, non‐myristoylated human Arf6 bound to GTPγS. Unlike their GDP‐bound forms, the active forms of Arf6 and Arf1 are very similar. Thus, the switch regions are discriminatory elements between Arf isoforms in their inactive but not in their active forms, a property that may generalize to other families of small G proteins. This suggests that GTP‐bound Arfs may establish specific interactions outside the switch regions and/or be recognized in their cellular context rather than as isolated proteins. The structure also allows further insight into the lack of spontaneous GTPase activity of Arf proteins.

Myosin VI Dimerization Triggers an Unfolding of a Three-Helix Bundle in Order to Extend Its Reach

Myosin VI challenges the prevailing theory of how myosin motors move on actin: the lever arm hypothesis. While the reverse directionality and large powerstroke of myosin VI can be attributed to unusual properties of a subdomain of the motor (converter with a unique insert), these adaptations cannot account for the large step size on actin. Either the lever arm hypothesis needs modification, or myosin VI has some unique form of extension of its lever arm. We determined the structure of the region immediately distal to the lever arm of the motor and show that it is a three-helix bundle. Based on C-terminal truncations that display the normal range of step sizes on actin, CD, fluorescence studies, and a partial deletion of the bundle, we demonstrate that this bundle unfolds upon dimerization of two myosin VI monomers. This unconventional mechanism generates an extension of the lever arm of myosin VI.

Structural Basis for Arf1-Mediated Recruitment of Arhgap21 to Golgi Membranes.

ARHGAP21 is a Rho family GTPase‐activating protein (RhoGAP) that controls the Arp2/3 complex and F‐actin dynamics at the Golgi complex by regulating the activity of the small GTPase Cdc42. ARHGAP21 is recruited to the Golgi by binding to another small GTPase, ARF1. Here, we present the crystal structure of the activated GTP‐bound form of ARF1 in a complex with the Arf‐binding domain (ArfBD) of ARHGAP21 at 2.1 Å resolution. We show that ArfBD comprises a PH domain adjoining a C‐terminal α helix, and that ARF1 interacts with both of these structural motifs through its switch regions and triggers structural rearrangement of the PH domain. We used site‐directed mutagenesis to confirm that both the PH domain and the helical motif are essential for the binding of ArfBD to ARF1 and for its recruitment to the Golgi. Our data demonstrate that two well‐known small GTPase‐binding motifs, the PH domain and the α helical motif, can combine to create a novel mode of binding to Arfs.

Structure of Arf6-Gdp Suggests a Basis for Guanine Nucleotide Exchange Factors Specificity

Arf6 is an isoform of Arf that localizes at the periphery of the cell where it has an essential role in endocytotic pathways. Its function does not overlap with that of Arf1, although the two proteins share ∼70% sequence identity and they have switch regions, whose conformation depends on the nature of the guanine nucleotide, with almost identical sequences. The crystal structure of Arf6–GDP at 2.3 A shows that it has a conformation similar to that of Arf1–GDP, which cannot bind membranes with high affinity. Significantly, the switch regions of Arf6 deviate by 2–5 A from those of Arf1. These differences are a consequence of the shorter N-terminal linker of Arf6 and of discrete sequence changes between Arf6 and Arf1. Mutational analysis shows that one of the positions which differs between Arf1 and Arf6 affects the configuration of the nucleotide binding site and thus the nucleotide binding properties of the Arf variant. Altogether, our results provide a structural basis for understanding how Arf1 and Arf6 can be distinguished by their guanine nucleotide exchange factors and suggest a model for the nucleotide/membrane cycle of Arf6.

The Structural Basis for the Large Powerstroke of Myosin VI

Due to a unique addition to the lever arm-positioning region (converter), class VI myosins move in the opposite direction (toward the minus-end of actin filaments) compared to other characterized myosin classes. However, the large size of the myosin VI lever arm swing (powerstroke) cannot be explained by our current view of the structural transitions that occur within the myosin motor. We have solved the crystal structure of a fragment of the myosin VI motor in the structural state that represents the starting point for movement on actin; the pre-powerstroke state. Unexpectedly, the converter itself rearranges to achieve a conformation that has not been seen for other myosins. This results in a much larger powerstroke than is achievable without the converter rearrangement. Moreover, it provides a new mechanism that could be exploited to increase the powerstroke of yet to be characterized plus-end-directed myosin classes.

Crystal structures of the small G protein Rap2A in complex with its substrate GTP, with GDP and with GTPgammaS.

The small G protein Rap2A has been crystallized in complex with GDP, GTP and GTPγS. The Rap2A–GTP complex is the first structure of a small G protein with its natural ligand GTP. It shows that the hydroxyl group of Tyr32 forms a hydrogen bond with the γ‐phosphate of GTP and with Gly13. This interaction does not exist in the Rap2A–GTPγS complex. Tyr32 is conserved in many small G proteins, which probably also form this hydrogen bond with GTP. In addition, Tyr32 is structurally equivalent to a conserved arginine that binds GTP in trimeric G proteins. The actual participation of Tyr32 in GTP hydrolysis is not yet clear, but several possible roles are discussed. The conformational changes between the GDP and GTP complexes are located essentially in the switch I and II regions as described for the related oncoprotein H‐Ras. However, the mobile segments vary in length and in the amplitude of movement. This suggests that even though similar regions might be involved in the GDP–GTP cycle of small G proteins, the details of the changes will be different for each G protein and will ensure the specificity of its interaction with a given set of cellular proteins.

The structural basis of Arf effector specificity: the crystal structure of ARF6 in a complex with JIP4

The JNK‐interacting proteins, JIP3 and JIP4, are specific effectors of the small GTP‐binding protein ARF6. The interaction of ARF6–GTP with the second leucine zipper (LZII) domains of JIP3/JIP4 regulates the binding of JIPs to kinesin‐1 and dynactin. Here, we report the crystal structure of ARF6–GTP bound to the JIP4‐LZII at 1.9 Å resolution. The complex is a heterotetramer with dyad symmetry arranged in an ARF6–(JIP4)2–ARF6 configuration. Comparison of the ARF6–JIP4 interface with the equivalent region of ARF1 shows the structural basis of JIP4s specificity for ARF6. Using site‐directed mutagenesis and surface plasmon resonance, we further show that non‐conserved residues at the switch region borders are the key structural determinants of JIP4 specificity. A structure‐derived model of the association of the ARF6–JIP3/JIP4 complex with membranes shows that the JIP4‐LZII coiled‐coil should lie along the membrane to prevent steric hindrances, resulting in only one ARF6 molecule bound. Such a heterotrimeric complex gives insights to better understand the ARF6‐mediated motor switch regulatory function.