Zhen Xu

Blood Systems Research Institute, San Francisco, California, United States

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Publications (10)45.28 Total impact

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    ABSTRACT: Kindlin-3 is a critical supporter of integrin function in platelets. Lack of expression of kindlin-3 protein in patients impairs integrin αIIbβ3-mediated platelet aggregation. Although kindlin-3 has been categorized as an integrin-binding partner, the functional significance of the direct interaction of kindlin-3 with integrin αIIbβ3 in platelets has not been established. Here, we evaluated the significance of the binding of kindlin-3 to integrin αIIbβ3 in platelets in supporting integrin αIIbβ3-mediated platelet functions.
    Arteriosclerosis Thrombosis and Vascular Biology 06/2014; · 6.34 Impact Factor
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    ABSTRACT: Kindlin family members are essential for supporting integrin activation by functionally cooperating with the talin head domain. Both the talin head and kindlin are FERM domain-containing proteins that can simultaneously interact with the integrin β cytoplasmic tails. While the talin head is well studied, the molecular basis of kindlin's interaction with integrin during integrin activation is still poorly understood. Here we defined the subdomain boundaries in kindlin-2 and evaluated their contribution to integrin activation and recognition. We found that each subdomain in kindlin-2 was obligatorily required for co-activating the integrin αIIbβ3 together with the talin head (inside-out signaling) and for enhancing integrin αIIbβ3-mediated cell spreading (outside-in signaling). To evaluate the involvement of the kindlin-2 subdomains in integrin interaction, we developed a FACS-based binding assay and found that an intact FERM domain in kindlin-2 was required for the interaction. Taking all together, these findings suggest that the integrity of kindlin-2 subdomains is prerequisite for supporting integrin recognition and for subsequent integrin activation.
    Biochemical and Biophysical Research Communications 04/2013; · 2.41 Impact Factor
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    ABSTRACT: Bag2, an atypical member of the Bag family of Hsp70 co-chaperones, acts as both an Hsp70 nucleotide-exchange factor and an inhibitor of the Hsp70-binding E3 ubiquitin ligase CHIP (carboxyl-terminus of Hsp70-interacting protein). The amino-terminal domain of Bag2 (Bag2-NTD), which is required for inhibition of CHIP, has no sequence homologs in the PDB. Native and selenomethionyl (SeMet) forms of Bag2-NTD were crystallized by hanging-drop vapor diffusion. Native Bag2-NTD crystals diffracted to 2.27 Å resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 75.5, b = 84.7, c = 114.1 Å. SeMet Bag2-NTD crystals diffracted to 3.10 Å resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 37.2, b = 53.3, c = 86.7 Å. Phases for the SeMet Bag2-NTD crystal were solved by single-wavelength anomalous diffraction. Initial phasing and model building using the 3.10 Å resolution SeMet Bag2-NTD data set suggested that Bag2-NTD forms a dimer and adopts a fold distinct from those of any domains annotated in the Pfam or SMART domain databases.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 06/2012; 68(Pt 6):647-51. · 0.55 Impact Factor
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    ABSTRACT: Both talin head domain and kindlin-2 interact with integrin β cytoplasmic tails, and they function in concert to induce integrin activation. Binding of talin head domain to β cytoplasmic tails has been characterized extensively, but information on the interaction of kindin-2 with this integrin segment is limited. In this study, we systematically examine the interactions of kindlin-2 with integrin β tails. Kindlin-2 interacted well with β(1) and β(3) tails but poorly with the β(2) cytoplasmic tail. This binding selectivity was determined by the non-conserved residues, primarily the three amino acids at the extreme C terminus of the β(3) tail, and the sequence in β(2) was non-permissive. The region at the C termini of integrin β(1) and β(3) tails recognized by kindlin-2 was a binding core of 12 amino acids. Kindlin-2 and talin head do not interact with one another but can bind simultaneously to the integrin β(3) tail without enhancing or inhibiting the interaction of the other binding partner. Kindlin-2 itself failed to directly unclasp integrin α/β tail complex, indicating that kindlin-2 must cooperate with talin to support the integrin activation mechanism.
    Journal of Biological Chemistry 05/2012; 287(29):24585-94. · 4.65 Impact Factor
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    ABSTRACT: Kindlins are a subclass of FERM-containing proteins that have recently emerged as key regulators of integrin receptor activation and signaling. As compared with the conventional FERM domain, the kindlin FERM domain contains an inserted pleckstrin homology (PH) domain that recognizes membrane phosphoinositides, including phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). Using NMR spectroscopy, we show that PIP3 site-specifically binds to kindlin-2 PH with substantial chemical shift changes that are much larger than PIP2. This suggests an enhanced association of kindlin-2 with membrane as mediated by PIP3 upon its conversion from PIP2 by phosphoinositide-3 kinase, a known regulator of integrin activation. We determined the NMR structure of the kindlin-2 PH domain bound to the head group of PIP3, inositol 1,3,4,5-tetraphosphate (IP4). The structure reveals a canonical PH domain fold, yet with a distinct IP4 binding pocket that appears highly conserved for the kindlin family members. Functional experiments demonstrate that although wild type kindlin-2 is capable of cooperating with integrin activator talin to induce synergistic integrin α(IIb)β(3) activation, this ability is significantly impaired for a phosphoinositide binding-defective kindlin-2 mutant. These results define a specific PIP3 recognition mode for the kindlin PH domain. Moreover, they shed light upon a mechanism as to how the PH domain mediates membrane engagement of kindlin-2 to promote its binding to integrin and cooperation with talin for regulation of integrin activation.
    Journal of Biological Chemistry 12/2011; 286(50):43334-42. · 4.65 Impact Factor
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    ABSTRACT: Mutations in the chloride channel cystic fibrosis transmembrane regulator (CFTR) cause cystic fibrosis, a genetic disorder characterized by defects in CFTR biosynthesis, localization to the cell surface, or activation by regulatory factors. It was discovered recently that surface localization of CFTR is stabilized by an interaction between the CFTR N terminus and the multidomain cytoskeletal protein filamin. The details of the CFTR-filamin interaction, however, are unclear. Using x-ray crystallography, we show how the CFTR N terminus binds to immunoglobulin-like repeat 21 of filamin A (FlnA-Ig21). CFTR binds to beta-strands C and D of FlnA-Ig21 using backbone-backbone hydrogen bonds, a linchpin serine residue, and hydrophobic side-chain packing. We use NMR to determine that the CFTR N terminus also binds to several other immunoglobulin-like repeats from filamin A in vitro. Our structural data explain why the cystic fibrosis-causing S13F mutation disrupts CFTR-filamin interaction. We show that FlnA-Ig repeats transfected into cultured Calu-3 cells disrupt CFTR-filamin interaction and reduce surface levels of CFTR. Our findings suggest that filamin A stabilizes surface CFTR by anchoring it to the actin cytoskeleton through interactions with multiple filamin Ig repeats. Such an interaction mode may allow filamins to cluster multiple CFTR molecules and to promote colocalization of CFTR and other filamin-binding proteins in the apical plasma membrane of epithelial cells.
    Journal of Biological Chemistry 03/2010; 285(22):17166-76. · 4.65 Impact Factor
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    ABSTRACT: Cochaperones are essential for Hsp70- and Hsc70-mediated folding of proteins and include nucleotide-exchange factors (NEFs) that assist protein folding by accelerating ADP-ATP exchange on Hsp70. The cochaperone Bag2 binds misfolded Hsp70 clients and also acts as an NEF, but the molecular basis for its function is unclear. We show that, rather than being a member of the Bag domain family, Bag2 contains a new type of Hsp70 NEF domain, which we call the 'brand new bag' (BNB) domain. Free and Hsc70-bound crystal structures of Bag2-BNB show its dimeric structure, in which a flanking linker helix and loop bind to Hsc70 to promote nucleotide exchange. NMR analysis demonstrates that the client binding sites and Hsc70-interaction sites of the Bag2-BNB overlap, and that Hsc70 can displace clients from Bag2-BNB, indicating a distinct mechanism for the regulation of Hsp70-mediated protein folding by Bag2.
    Nature Structural & Molecular Biology 12/2008; 15(12):1309-17. · 11.90 Impact Factor
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    ABSTRACT: Myotrophin, a 12-kDa ankyrin repeat protein, stimulates protein synthesis and cardiomyocyte growth to initiate cardiac hypertrophy by activating the NF-κB signaling cascade. We found that, after internalization into myocytes, myotrophin cotranslocates into the nucleus with p65 to stimulate myocyte growth. We used structure-based mutations on the hairpin loops of myotrophin to determine the effect of the loops on myotrophin and p65 localization, induction of protein synthesis, and cardiac hypertrophy. Loop mutants, most prominently glutamic acid 33→alanine (E33A), stimulated protein synthesis much less than wild type. Myotrophin-E33A internalized into myocytes but did not translocate into the nucleus and failed to promote nuclear translocation of p65. In addition, two cardiac hypertrophy marker genes, atrial natriuretic factor and β-myosin heavy chain, were not up-regulated in E33A-treated cells. Myotrophin-induced myocyte growth and initiation of hypertrophy thus require nuclear co-translocation of myotrophin and p65, in a manner that depends crucially on the myotrophin hairpin loops.
    Journal of Biological Chemistry 10/2008; 283(41):27947-27956. · 4.65 Impact Factor
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    ABSTRACT: Ubiquitin (E3) ligases interact with specific ubiquitin conjugating (E2) enzymes to ubiquitinate particular substrate proteins. As the combination of E2 and E3 dictates the type and biological consequence of ubiquitination, it is important to understand the basis of specificity in E2:E3 interactions. The E3 ligase CHIP interacts with Hsp70 and Hsp90 and ubiquitinates client proteins that are chaperoned by these heat shock proteins. CHIP interacts with two types of E2 enzymes, UbcH5 and Ubc13-Uev1a. It is unclear, however, why CHIP binds these E2 enzymes rather than others, and whether CHIP interacts preferentially with UbcH5 or Ubc13-Uev1a, which form different types of polyubiquitin chains. The 2.9 A crystal structure of the CHIP U-box domain complexed with UbcH5a shows that CHIP binds to UbcH5 and Ubc13 through similar specificity determinants, including a key S-P-A motif on the E2 enzymes. The determinants make different relative contributions to the overall interactions between CHIP and the two E2 enzymes. CHIP undergoes auto-ubiquitination by UbcH5 but not by Ubc13-Uev1a. Instead, CHIP drives the formation of unanchored polyubiquitin by Ubc13-Uev1a. CHIP also interacts productively with the class III E2 enzyme Ube2e2, in which the UbcH5- and Ubc13-binding specificity determinants are highly conserved. The CHIP:UbcH5a structure emphasizes the importance of specificity determinants located on the long loops and central helix of the CHIP U-box, and on the N-terminal helix and loops L4 and L7 of its cognate E2 enzymes. The S-P-A motif and other specificity determinants define the set of cognate E2 enzymes for CHIP, which likely includes several Class III E2 enzymes. CHIP's interactions with UbcH5, Ube2e2 and Ubc13-Uev1a are consistent with the notion that Ubc13-Uev1a may work sequentially with other E2 enzymes to carry out K63-linked polyubiquitination of CHIP substrates.
    BMC Structural Biology 02/2008; 8:26. · 2.10 Impact Factor
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    ABSTRACT: The heat-shock proteins Hsp70 and Hsp90 play a crucial role in regulating protein quality control both by refolding and by preventing the aggregation of misfolded proteins. It has recently been shown that Hsp70 and Hsp90 act not only in protein refolding but also cooperate with the C terminus of Hsp70 interacting protein (CHIP), a multidomain ubiquitin ligase, to mediate the degradation of unfolded proteins. We present the crystal structure of the helical linker domain and U-box domain of zebrafish CHIP (DrCHIP-HU). The structure of DrCHIP-HU shows a symmetric homodimer. The conformation of the helical linker domains and the relative positions of the helical and U-box domains differ substantially in DrCHIP-HU from those in a recently published structure of an asymmetric dimer of mammalian (mouse) CHIP. We used an in vitro ubiquitination assay to identify residues, located on two long loops and a central alpha helix of the CHIP U-box domain, that are important for interacting with the ubiquitin-conjugating enzyme UbcH5b. In addition, we used NMR spectroscopy to define a complementary interaction surface located on the N-terminal alpha helix and the L4 and L7 loops of UbcH5b. Our results provide insights into conformational variability in the domain arrangement of CHIP and into U-box-mediated recruitment of UbcH5b for the ubiquitination of Hsp70 and Hsp90 substrates.
    Biochemistry 05/2006; 45(15):4749-59. · 3.38 Impact Factor