Processive steps in the reverse direction require uncoupling of the lead head lever arm of myosin VI.

Myosin VI is the only known reverse-direction myosin motor. It has an unprecedented means of amplifying movements within the motor involving rearrangements of the converter subdomain at the C terminus of the motor and an unusual lever arm projecting from the converter. While the average step size of a myosin VI dimer is 30-36 nm, the step size is highly variable, presenting a challenge to the lever arm mechanism by which all myosins are thought to move. Herein, we present structures of myosin VI that reveal regions of compliance that allow an uncoupling of the lead head when movement is modeled on actin. The location of the compliance restricts the possible actin binding sites and predicts the observed stepping behavior. The model reveals that myosin VI, unlike plus-end directed myosins, does not use a pure lever arm mechanism, but instead steps with a mechanism analogous to the kinesin neck-linker uncoupling model.

Characterization of the binding mode of JNK-interacting protein 1 (JIP1) to kinesin-light chain 1 (KLC1).

JIP1 was first identified as scaffold protein for the MAP kinase JNK and is a cargo protein for the kinesin1 molecular motor. JIP1 plays significant and broad roles in neurons, mainly as a regulator of kinesin1-dependent transport, and is associated with human pathologies such as cancer and Alzheimer disease. JIP1 is specifically recruited by the kinesin-light chain 1 (KLC1) of kinesin1, but the details of this interaction are not yet fully elucidated. Here, using calorimetry, we extensively biochemically characterized the interaction between KLC1 and JIP1. Using various truncated fragments of the tetratricopeptide repeat (TPR) domain of KLC1, we narrowed down its JIP1-binding region and identified seven KLC1 residues critical for JIP1 binding. These isothermal titration calorimetry (ITC)-based binding data enabled us to footprint the JIP1-binding site on KLC1-TPR. This footprint was used to uncover the structural basis for the marginal inhibition of JIP1 binding by the autoinhibitory LFP-acidic motif of KLC1, as well as for the competition between JIP1 and another cargo protein of kinesin1, the W-acidic motif-containing alcadein-α. Also, we examined the role of each of these critical residues of KLC1 for JIP1 binding in light of the previously reported crystal structure of the KLC1-TPR:JIP1 complex. Finally, sequence search in eukaryotic genomes identified several proteins, among which is SH2D6, that exhibit a motif similar to the KLC1-binding motif of JIP1. Overall, our extensive biochemical characterization of the KLC:JIP1 interaction, as well as identification of potential KLC1-binding partners, improves the understanding of how this growing family of cargos is recruited to kinesin1 by KLC1.

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.

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 A 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 JIP4's 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.

The post-rigor structure of myosin VI and implications for the recovery stroke.

Myosin VI has an unexpectedly large swing of its lever arm (powerstroke) that optimizes its unique reverse direction movement. The basis for this is an unprecedented rearrangement of the subdomain to which the lever arm is attached, referred to as the converter. It is unclear at what point(s) in the myosin VI ATPase cycle rearrangements in the converter occur, and how this would effect lever arm position. We solved the structure of myosin VI with an ATP analogue (ADP.BeF3) bound in its nucleotide-binding pocket. The structure reveals that no rearrangement in the converter occur upon ATP binding. Based on previously solved myosin structures, our structure suggests that no reversal of the powerstroke occurs during detachment of myosin VI from actin. The structure also reveals novel features of the myosin VI motor that may be important in maintaining the converter conformation during detachment from actin, and other features that may promote rapid rearrangements in the structure following actin detachment that enable hydrolysis of ATP.

Structure of the GDP-bound G domain of the RGK protein Rem2.

RGK proteins are atypical small GTP-binding proteins that are involved in the regulation of voltage-dependent calcium channels and actin cytoskeleton remodelling. The structure of the Rem2 G domain bound to GDP is reported here in a monoclinic crystal form at 2.66 Å resolution. It is very similar to the structure determined previously from an orthorhombic crystal form. However, differences in the crystal-packing environment revealed that the switch I and switch II regions are flexible and not ordered as previously reported. Comparison of the available RGK protein structures along with those of other small GTP-binding proteins highlights two structural features characteristic of this atypical family and suggests that the conserved tryptophan residue in the DXWEX motif may be a structural determinant of the nucleotide-binding affinity.

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

Here we solve a 2.4-A 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.

Structural snapshots of the kinesin-2 OSM-3 along its nucleotide cycle: implications for the ATP hydrolysis mechanism.

Motile kinesins are motor proteins that translocate along microtubules as they hydrolyze ATP. They share a conserved motor domain which harbors both ATPase and microtubule-binding activities. An ATP hydrolysis mechanism involving two water molecules has been proposed based on the structure of the kinesin-5 Eg5 bound to an ATP analog. Whether this mechanism is general in the kinesin superfamily remains uncertain. Here, we present structural snapshots of the motor domain of OSM-3 along its nucleotide cycle. OSM-3 belongs to the homodimeric kinesin-2 subfamily and is the Caenorhabditis elegans homologue of human KIF17. OSM-3 bound to ADP or devoid of a nucleotide shows features of ADP-kinesins with a docked neck linker. When bound to an ATP analog, OSM-3 adopts a conformation similar to those of several ATP-like kinesins, either isolated or bound to tubulin. Moreover, the OSM-3 nucleotide-binding site is virtually identical to that of ATP-like Eg5, demonstrating a shared ATPase mechanism. Therefore, our data extend to kinesin-2 the two-water ATP hydrolysis mechanism and further suggest that it is universal within the kinesin superfamily. PROTEIN DATABASE ENTRIES: 7A3Z, 7A40, 7A5E.

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 A 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.

Structural characterization of the RH1-LZI tandem of JIP3/4 highlights RH1 domains as a cytoskeletal motor-binding motif.

JIP3 and JIP4 (JNK-interacting proteins 3 and 4) are adaptors for cargo recruitment by dynein/dynactin and kinesin1 motors. Both are dimers that are stabilised by two sections of leucine zipper coiled coils. The N-terminal Leucine Zipper I (LZI) belongs to a section that binds dynein-DLIC and kinesin1-KHC, whilst the medial Leucine Zipper II (LZII) binds dynactin-p150glued and kinesin1-KLC. Structural data is available for the LZII, but the LZI section is still uncharacterized. Here we characterize the N-terminal part of JIP3/4 which consists of an RH1 (RILP homology 1) domain followed by the LZI coiled coil using bioinformatical, biophysical and structural approaches. The RH1-LZI tandem of JIP3 associates as a high affinity homodimer exhibiting elongated alpha-helical fold. 3D homology modelling of the RH1-LZI tandem reveals that the kinesin1-KHC binding site mainly overlaps with the RH1 domain. A sequence comparison search indicates that only one other protein family has RH1 domains similar to those of JIP3/4, the RILP (Rab-interacting lysosomal protein) family which consists of adaptor proteins linking Rab GTPases to cytoskeletal motors. RILPL2 is recruited through its RH1 domain by the myosin 5a motor. Here, we showed that the RH1 domain of JIP3 also interacts with myosin 5 A in vitro, highlighting JIP3/4 as possible myosin 5a adaptors. Finally, we propose that JIP3/4 and RILP family members define a unique RH1/RH2-architecture adaptor superfamily linking cytoskeletal motors and Rab GTPases.

Correction: Structural plasticity of the N-terminal capping helix of the TPR domain of kinesin light chain.

[This corrects the article DOI: 10.1371/journal.pone.0186354.].

Structural plasticity of the N-terminal capping helix of the TPR domain of kinesin light chain.

Kinesin1 plays a major role in neuronal transport by recruiting many different cargos through its kinesin light chain (KLC). Various structurally unrelated cargos interact with the conserved tetratricopeptide repeat (TPR) domain of KLC. The N-terminal capping helix of the TPR domain exhibits an atypical sequence and structural features that may contribute to the versatility of the TPR domain to bind different cargos. We determined crystal structures of the TPR domain of both KLC1 and KLC2 encompassing the N-terminal capping helix and show that this helix exhibits two distinct and defined orientations relative to the rest of the TPR domain. Such a difference in orientation gives rise, at the N-terminal part of the groove, to the formation of one hydrophobic pocket, as well as to electrostatic variations at the groove surface. We present a comprehensive structural analysis of available KLC1/2-TPR domain structures that highlights that ligand binding into the groove can be specific of one or the other N-terminal capping helix orientations. Further, structural analysis reveals that the N-terminal capping helix is always involved in crystal packing contacts, especially in a TPR1:TPR1' contact which highlights its propensity to be a protein-protein interaction site. Together, these results underline that the structural plasticity of the N-terminal capping helix might represent a structural determinant for TPR domain structural versatility in cargo binding.

Structure of a truncated form of leucine zipper II of JIP3 reveals an unexpected antiparallel coiled-coil arrangement.

JIP3 and JIP4, two highly related scaffolding proteins for MAP kinases, are binding partners for two molecular motors as well as for the small G protein ARF6. The leucine zipper II (LZII) region of JIP3/4 is the binding site for these three partners. Previously, the crystal structure of ARF6 bound to JIP4 revealed LZII in a parallel coiled-coil arrangement. Here, the crystal structure of an N-terminally truncated form of LZII of JIP3 alone shows an unexpected antiparallel arrangement. Using molecular dynamics and modelling, the stability of this antiparallel LZII arrangement, as well as its specificity for ARF6, were investigated. This study highlights that N-terminal truncation of LZII can change its coiled-coil orientation without affecting its overall stability. Further, a conserved buried asparagine residue was pinpointed as a possible structural determinant for this dramatic structural rearrangement. Thus, LZII of JIP3/4 is a versatile structural motif, modifications of which can impact partner recognition and thus biological function.

Structural features common to intracellularly acting toxins from bacteria.

This mini-review focuses on structural features shared by bacterial intracellularly-acting toxins. These complex proteins adopt an A(n)B(m) assembly. B(m) is a cellular-uptake machinery that delivers the enzymatic A(n) component, where it specifically modifies an intracellular eukaryotic cell target. In this nomenclature, the m index reflects the mono- or oligomeric (homo or hetero) state of the B component and the n index indicates the number of A molecules that concomitantly bind to B(m). A structural analysis of the available 3D structures suggests that each of the A molecules that constitute the A(n) component can be divided into A(link) and A(enz) sub-domains, with A(link) specifically linking the enzymatically active A(enz) domain to a given B(m). This module-based A(n)B(m) assembly seems decisive for natural intracellularly-acting toxins to be potent and for the success of engineered toxins.

Structural biology of Arf and Rab GTPases' effector recruitment and specificity.

Arf and Rab proteins, members of small GTPases superfamily, localize to specific subcellular compartments and regulate intracellular trafficking. To carry out their cellular functions, Arfs/Rabs interact with numerous and structurally diverse effector proteins. Over the years, a number of Arf/Rab:effector complexes have been crystallized and their structures reveal shared binding modes including α-helical packing, ß-ß complementation, and heterotetrameric assemblies. We review available structural information and provide a framework for in-depth analysis of complexes. The unifying features that we identify are organized into a classification scheme for different modes of Arf/Rab:effector interactions, which includes "all-α-helical," "mixed α-helical," "ß-ß zipping," and "bivalent" modes of binding. Additionally, we highlight structural determinants that are the basis of effector specificity. We conclude by expanding on functional implications that are emerging from available structural information under our proposed classification scheme.

Structural organisation of the rotavirus nonstructural protein NSP5.

Rotavirus is one of the leading agents of gastroenteritis worldwide. During infection, viral factories (viroplasms) are formed. The rotavirus nonstructural proteins NSP5 and NSP2 are the major building blocks of viroplasms; however, NSP5 function and organisation remain elusive. In this report, we present a structural characterisation of NSP5. Multi-angle laser light scattering, sedimentation velocity and equilibrium sedimentation experiments demonstrate that recombinant full-length NSP5 forms a decamer in solution. Far-Western, pull-down and multi-angle laser light scattering experiments show that NSP5 has two oligomerisation regions. The first region, residues 103-146, is involved in NSP5 dimerisation, whereas the second region, residues 189-198, is responsible for NSP5 decamerisation. Circular dichroism analyses of full-length and truncated forms of NSP5 reveal that the decamerisation region is helical, whereas the dimerisation region involves ß-sheets. From these circular dichroism experiments, we also show that the NSP5 protomers contain two α-helices, a disordered N-terminal half and a C-terminal half that is primarily composed of ß-sheet folds. This extensive structural characterisation of NSP5 led us to propose a model for its quaternary organisation. Finally, co-expression of NSP5 fragments and NSP2 in uninfected cells shows that the NSP5 decamerisation region is required for viroplasm-like structure formation. However, in vitro, the NSP5 decamerisation region partially inhibits the NSP2-NSP5 interaction. Our NSP5 model suggests that steric hindrance prevents NSP2 from binding to all NSP5 protomers. Some protomers may thus be free to interact with other NSP5 binding partners, such as viral RNAs and the viral polymerase VP1, to perform functions other than viroplasm organisation.

Toward a structural understanding of arf family:effector specificity.

Arf family proteins are critical regulators of intracellular trafficking and actin cytoskeleton dynamics. To carry out their cellular functions, Arf family proteins interact with various effectors that differ in nature and structure. Understanding how these proteins interact with structurally different partners and are distinguished by specific effectors while being closely related requires a structural characterization and comparison of the various Arf family:effector complexes. Recent structural reports of Arf and Arl proteins in complex with different downstream effectors shed new light on general and specific structural recognition determinants characteristic of Arf family proteins.

RAB6C is a retrogene that encodes a centrosomal protein involved in cell cycle progression.

Rab-GTPases are key regulators of membrane transport, and growing evidence indicates that their expression levels are altered in certain human malignancies, including cancer. Rab6C, a newly identified Rab6 subfamily member, has attracted recent attention because its reduced expression might confer a selective advantage to drug-resistant breast cancer cells. Here, we report that RAB6C is a primate-specific retrogene derived from a RAB6A' transcript. RAB6C is transcribed in a limited number of human tissues including brain, testis, prostate, and breast. Endogenous Rab6C is considerably less abundant and has a much shorter half-life than Rab6A'. Comparison of the GTP-binding motifs of Rab6C and Rab6A', homology modeling, and GTP-blot overlay assays indicate that amino acid changes in Rab6C have greatly reduced its GTP-binding affinity. Instead, the noncanonical GTP-binding domain of Rab6C mediates localization of the protein to the centrosome. Overexpression of Rab6C results in G1 arrest, and its specific depletion generates tetraploid cells with supernumerary centrosomes, revealing a role of Rab6C in events related to the centrosome and cell cycle progression. Thus, RAB6C is a rare example of a recently emerged retrogene that has acquired the status of a new gene, encoding a functional protein with altered characteristics compared to Rab6A'.

Structural basis for the NAD-hydrolysis mechanism and the ARTT-loop plasticity of C3 exoenzymes.

C3-like exoenzymes are ADP-ribosyltransferases that specifically modify some Rho GTPase proteins, leading to their sequestration in the cytoplasm, and thus inhibiting their regulatory activity on the actin cytoskeleton. This modification process goes through three sequential steps involving NAD-hydrolysis, Rho recognition, and binding, leading to Rho ADP-ribosylation. Independently, three distinct residues within the ARTT loop of the C3 exoenzymes are critical for each of these steps. Supporting the critical role of the ARTT loop, we have shown previously that it adopts a distinct conformation upon NAD binding. Here, we present seven wild-type and ARTT loop-mutant structures of C3 exoenzyme of Clostridium botulinum free and bound to its true substrate, NAD, and to its NAD-hydrolysis product, nicotinamide. Altogether, these structures expand our understanding of the conformational diversity of the C3 exoenzyme, mainly within the ARTT loop.