yu Wen

Myosin VI Undergoes Cargo-Mediated Dimerization

Myosin VI is the only known molecular motor that moves toward the minus ends of actin filaments; thus, it plays unique roles in diverse cellular processes. The processive walking of myosin VI on actin filaments requires dimerization of the motor, but the protein can also function as a nonprocessive monomer. The molecular mechanism governing the monomer-dimer conversion is not clear. We report the high-resolution NMR structure of the cargo-free myosin VI cargo-binding domain (CBD) and show that it is a stable monomer in solution. The myosin VI CBD binds to a fragment of the clathrin-coated vesicle adaptor Dab2 with a high affinity, and the X-ray structure of the myosin VI CBD in complex with Dab2 reveals that the motor undergoes a cargo-binding-mediated dimerization. The cargo-binding-induced dimerization may represent a general paradigm for the regulation of processivity for myosin VI as well as other myosins, including myosin VII and myosin X.

LGN/mInsc and LGN/NuMA Complex Structures Suggest Distinct Functions in Asymmetric Cell Division for the Par3/mInsc/LGN and Gαi/LGN/NuMA Pathways

Asymmetric cell division requires the establishment of cortical cell polarity and the orientation of the mitotic spindle along the axis of cell polarity. Evidence from invertebrates demonstrates that the Par3/Par6/aPKC and NuMA/LGN/Gαi complexes, which are thought to be physically linked by the adaptor protein mInscuteable (mInsc), play indispensable roles in this process. However, the molecular basis for the binding of LGN to NuMA and mInsc is poorly understood. The high-resolution structures of the LGN/NuMA and LGN/mInsc complexes presented here provide mechanistic insights into the distinct and highly specific interactions of the LGN TPRs with mInsc and NuMA. Structural comparisons, together with biochemical and cell biology studies, demonstrate that the interactions of NuMA and mInsc with LGN are mutually exclusive, with mInsc binding preferentially. Our results suggest that the Par3/mInsc/LGN and NuMA/LGN/Gαi complexes play sequential and partially overlapping roles in asymmetric cell division.

Guanylate kinase domains of the MAGUK family scaffold proteins as specific phospho-protein-binding modules

Membrane‐associated guanylate kinases (MAGUKs) are a large family of scaffold proteins that play essential roles in tissue developments, cell–cell communications, cell polarity control, and cellular signal transductions. Despite extensive studies over the past two decades, the functions of the signature guanylate kinase domain (GK) of MAGUKs are poorly understood. Here we show that the GK domain of DLG1/SAP97 binds to asymmetric cell division regulatory protein LGN in a phosphorylation‐dependent manner. The structure of the DLG1 SH3‐GK tandem in complex with a phospho‐LGN peptide reveals that the GMP‐binding site of GK has evolved into a specific pSer/pThr‐binding pocket. Residues both N‐ and C‐terminal to the pSer are also critical for the specific binding of the phospho‐LGN peptide to GK. We further demonstrate that the previously reported GK domain‐mediated interactions of DLGs with other targets, such as GKAP/DLGAP1/SAPAP1 and SPAR, are also phosphorylation dependent. Finally, we provide evidence that other MAGUK GKs also function as phospho‐peptide‐binding modules. The discovery of the phosphorylation‐dependent MAGUK GK/target interactions indicates that MAGUK scaffold‐mediated signalling complex organizations are dynamically regulated.

Autoinhibition of UNC5b Revealed by the Cytoplasmic Domain Structure of the Receptor

The cytoplasmic domains of UNC5 are responsible for its netrin-mediated signaling events in axonal migrations, blood vessel patterning, and apoptosis, although the molecular mechanisms governing these processes are unknown. To provide a foundation for the elucidation of the UNC5-mediated signaling mechanism, we determined the crystal structure of the cytoplasmic portion of UNC5b. We found that it contains three distinctly folded domains, namely ZU5, UPA, and death domain (DD). These three domains form a structural supramodule, with ZU5 binding to both UPA and DD, thereby locking the ZU5-UPA-DD supramodule in a closed conformation and suppressing its biological activities. Release of the closed conformation of the ZU5-UPA-DD supramodule leads to the activation of the receptor in the promotion of apoptosis and blood vessel patterning. Finally, we provide evidence showing that the supramodular nature of UNC5 ZU5-UPA-DD is likely to be shared by the ankyrin and PIDD families of scaffold proteins.

Structure of the split PH domain and distinct lipid-binding properties of the PH–PDZ supramodule of α-syntrophin

Pleckstrin homology (PH) domains play diverse roles in cytoskeletal dynamics and signal transduction. Split PH domains represent a unique subclass of PH domains that have been implicated in interactions with complementary partial PH domains ‘hidden’ in many proteins. Whether partial PH domains exist as independent structural units alone and whether two halves of a split PH domain can fold together to form an intact PH domain are not known. Here, we solved the structure of the PHN–PDZ–PHC tandem of α‐syntrophin. The split PH domain of α‐syntrophin adopts a canonical PH domain fold. The isolated partial PH domains of α‐syntrophin, although completely unfolded, remain soluble in solution. Mixing of the two isolated domains induces de novo folding and yields a stable PH domain. Our results demonstrate that two complementary partial PH domains are capable of binding to each other to form an intact PH domain. We further showed that the PHN–PDZ–PHC tandem forms a functionally distinct supramodule, in which the split PH domain and the PDZ domain function synergistically in binding to inositol phospholipids.

Serine 88 Phosphorylation of the 8-kDa Dynein Light Chain 1 Is a Molecular Switch for Its Dimerization Status and Functions

Dynein light chain 1 (DLC1, also known as DYNLL1, LC8, and PIN), a ubiquitously expressed and highly conserved protein, participates in a variety of essential intracellular events. Transition of DLC1 between dimer and monomer forms might play a crucial role in its function. However, the molecular mechanism(s) that control the transition remain unknown. DLC1 phosphorylation on Ser88 by p21-activated kinase 1 (Pak1), a signaling nodule, promotes mammalian cell survival by regulating its interaction with Bim and the stability of Bim. Here we discovered that phosphorylation of Ser88, which juxtapose each other at the interface of the DLC dimer, disrupts DLC1 dimer formation and consequently impairs its interaction with Bim. Overexpression of a Ser88 phosphorylation-inactive DLC1 mutant in mammary epithelium cells and in a transgenic animal model caused apoptosis and accelerated mammary gland involution, respectively, with increased Bim levels. Structural and biophysical studies suggested that phosphorylation-mimicking mutation leads to dissociation of the DLC1 dimer to a pure folded monomer. The phosphorylation-induced DLC1 monomer is incapable of binding to its substrate Bim. These findings reveal a previously unrecognized regulatory mechanism of DLC1 in which the Ser88 phosphorylation acts as a molecular switch for the transition of DLC1 from dimer to monomer, thereby modulating its interaction with substrates and consequently regulating the functions of DLC1.

Cdc42‐dependent formation of the ZO‐1/MRCKβ complex at the leading edge controls cell migration

Zonula occludens (ZO)‐1 is a multi‐domain scaffold protein known to have critical roles in the establishment of cell–cell adhesions and the maintenance of stable tissue structures through the targeting, anchoring, and clustering of transmembrane adhesion molecules and cytoskeletal proteins. Here, we report that ZO‐1 directly binds to MRCKβ, a Cdc42 effector kinase that modulates cell protrusion and migration, at the leading edge of migrating cells. Structural studies reveal that the binding of a β hairpin from GRINL1A converts ZO‐1 ZU5 into a complete ZU5‐fold. A similar interaction mode is likely to occur between ZO‐1 ZU5 and MRCKβ. The interaction between ZO‐1 and MRCKβ requires the kinase to be primed by Cdc42 due to the closed conformation of the kinase. Formation of the ZO‐1/MRCKβ complex enriches the kinase at the lamellae of migrating cells. Disruption of the ZO‐1/MRCKβ complex inhibits MRCKβ‐mediated cell migration. These results demonstrate that ZO‐1, a classical scaffold protein with accepted roles in maintaining cell–cell adhesions in stable tissues, also has an active role in cell migration during processes such as tissue development and remodelling.

Structure Basis and Unconventional Lipid Membrane Binding Properties of the PH-C1 Tandem of Rho Kinases *□

Rho kinase (ROCK), a downstream effector of Rho GTPase, is a serine/threonine protein kinase that regulates many crucial cellular processes via control of cytoskeletal structures. The C-terminal PH-C1 tandem of ROCKs has been implicated to play an autoinhibitory role by sequestering the N-terminal kinase domain and reducing its kinase activity. The binding of lipids to the pleckstrin homology (PH) domain not only regulates the localization of the protein but also releases the kinase domain from the close conformation and thereby activates its kinase activity. However, the molecular mechanism governing the ROCK PH-C1 tandem-mediated lipid membrane interaction is not known. In this study, we demonstrate that ROCK is a new member of the split PH domain family of proteins. The ROCK split PH domain folds into a canonical PH domain structure. The insertion of the atypical C1 domain in the middle does not alter the structure of the PH domain. We further show that the C1 domain of ROCK lacks the diacylglycerol/phorbol ester binding pocket seen in other canonical C1 domains. Instead, the inserted C1 domain and the PH domain function cooperatively in binding to membrane bilayers via the unconventional positively charged surfaces on each domain. Finally, the analysis of all split PH domains with known structures indicates that split PH domains represent a unique class of tandem protein modules, each possessing distinct structural and functional features.

Phosphorylation of DCC by ERK2 Is Facilitated by Direct Docking of the Receptor P1 Domain to the Kinase

Netrin receptor DCC plays critical roles in many cellular processes, including axonal outgrowth and migration, angiogenesis, and apoptosis, but the molecular basis of DCC-mediated signaling is largely unclear. ERK2, a member of the MAPK family, is one of the few proteins known to be involved in DCC-mediated signaling. Here, we report that ERK2 directly interacts with DCC, and the ERK2-binding region was mapped to the conserved intracellular P1 domain of the receptor. The structure of ERK2 in complex with the P1 domain of DCC reveals that DCC contains a MAPK docking motif. The docking of the P1 domain onto ERK2 physically positions several phosphorylation sites of DCC in the vicinity of the kinase active site. We further show that the docking interaction between the P1 domain and ERK2 is essential for the ERK2-mediated phosphorylation of DCC. We conclude that DCC signaling is directly coupled with MAPK signaling cascades.

Lipid-Induced Conformational Switch Controls Fusion Activity of Longin Domain SNARE Ykt6

While most SNAREs are permanently anchored to membranes by their transmembrane domains, the dually lipidated SNARE Ykt6 is found both on intracellular membranes and in the cytosol. The cytosolic Ykt6 is inactive due to the autoinhibition of the SNARE core by its longin domain, although the molecular basis of this inhibition is unknown. Here, we demonstrate that unlipidated Ykt6 adopts multiple conformations, with a small population in the closed state. The structure of Ykt6 in complex with a fatty acid suggests that, upon farnesylation, the Ykt6 SNARE core forms four alpha helices that wrap around the longin domain, forming a dominantly closed conformation. The fatty acid, buried in a hydrophobic groove formed between the longin domain and its SNARE core, is essential for maintaining the autoinhibited conformation of Ykt6. Our study reveals that the posttranslationally attached farnesyl group can actively regulate Ykt6 fusion activity in addition to its anticipated membrane-anchoring role.