Structure-Function Analysis of the Auxilin J-Domain Reveals an Extended Hsc70 Interaction Interface ²,‡ Jianwen Jiang, Alexander B. Taylor, Kondury Prasad, Yumiko Ishikawa-Brush, P. John Hart, Eileen M. Lafer, and Rui Sousa* Department of Biochemistry, UniVersity of Texas Health Science Center, 7703 Floyd Curl DriVe, San Antonio, Texas 78229-3900 ReceiVed February 17, 2003; ReVised Manuscript ReceiVed March 26, 2003 ABSTRACT: J-domains are widespread protein interaction modules involved in recruiting and stimulating the activity of Hsp70 family chaperones. We have determined the crystal structure of the J-domain of auxilin, a protein which is involved in uncoating clathrin-coated vesicles. Comparison to the known structures of J-domains from four other proteins reveals that the auxilin J-domain is the most divergent of all J-domain structures described to date. In addition to the canonical J-domain features described previously, the auxilin J-domain contains an extra N-terminal helix and a long loop inserted between helices I and II. The latter loop extends the positively charged surface which forms the Hsc70 binding site, and is shown by directed mutagenesis and surface plasmon resonance to contain side chains important for binding to Hsc70. The J-domain is a widespread protein interaction module, conserved from bacteria to humans, and involved in recruit- ing Hsp70 family chaperone proteins to their sites of action (1, 2). J-domain proteins include Escherichia coli Hsp40 and its prokaryotic and eukaryotic orthologues, as well as proteins which typically contain a J-domain as well as a substrate binding domain but lack the gly-phe-rich domain and cys- rich zinc finger characteristic of the Hsp40 co-chaperones (2). The substrate binding domains of J-domain proteins function by binding proteins which are then presented to Hsp70 via interactions between Hsp70 and the J-domain. Different J-domain proteins contain distinct substrate binding domains, and it is these domains which, by targeting Hsp70s to specific proteins or protein complexes, confer much of the specificity for the different roles played by Hsp70s in the cell. These include prevention of protein aggregation, facilitation of folding, protein translocation, disassembly of protein complexes, proteolytic facilitation, and even control of regulatory protein activity (1, 2). One of the best understood of these reactions is the clathrin coat disassembly reaction mediated by Hsc70 (3) and auxilin (4-9). Auxilin is a 910-residue protein composed of an N-terminal cys-rich tensin-like domain, a central clathrin binding domain, and a C-terminal J-domain. Auxilin binds to clathrin-coated vesicles through its clathrin binding domain and then recruits Hsc70 via its J-domain. In a reaction coupled to ATP hydrolysis, auxilin and Hsc70 disassemble the clathrin coat to form a transport vesicle. The location of the J-domain at the C-terminus of auxilin is unusual in that most J-domain proteins, and all Hsp40 orthologues, have the J-domain at the N-terminus (2). The structures of three J-do- mains (from E. coli and human Hsp40, and from polyoma virus T-Ag) have been solved by NMR, and one J-domain protein structure (E. coli Hsp20) has been solved by X-ray crystallography (10-13). Despite limited sequence similarity, all four of these structures exhibit canonical 75-79-residue J-domain structures comprising a similar arrangement of four R helices, with helices II and III forming an antiparallel hairpin which contains the invariant HPD J-domain signature motif in the loop between these helices. Here we describe the crystal structure of an auxilin C-ter- minal fragment which corresponds to this protein’s J-domain. We find that, relative to the canonical J-domain structure, the auxilin J-domain is distinct: it contains an additional N-terminal helix and a long loop inserted between helices I and II. Directed mutagenesis and surface plasmon resonance experiments show that this loop forms part of the functional interface for interaction with Hsc70. MATERIALS AND METHODS Expression and Purification of Selenomethionine-Labeled J-Domain. Competent E. coli strain B834(DE3) was trans- formed with a plasmid which expresses bovine auxilin resi- dues 810-910 as part of a GST fusion protein (5). Trans- formed cells were grown in 12 L of M9 media supplemented with selenomethionine to an OD of 0.4-0.7 at 37 °C, and expression of the fusion protein was induced by addition of IPTG to 1 mM. Cells were grown for an additional 3-6 h, harvested by centrifugation, and resuspended in 150 mL of 50 mM Tris pH 8.0, 1 mM DTT, 1 mM EDTA, 1 mM PMSF, and 5% glycerol. Cells were lysed by sonication and 0.05 vol of 10% Polymin P, pH 8.0, was added to precipitate nucleic acid. The lysate was cleared by centrifugation at 15 000 rpm for 15 min, and the supernatant was loaded onto 100 mL of glutathione resin (Pharmacia). The resin was ² Supported by NIH grants NS29051 (to E.M.L.) and GM52522 (to R.S.), Welch Grant AQ-1399 (to P.J.H.), and NIH shared instrumenta- tion grant 1S10RR15883. ‡ Coordinates deposited in PDB 1NZ6. * To whom correspondence should be addressed. Phone: (210) 567- 8782. Fax: (210) 567-8778. E-mail: sousa@biochem.uthscsa.edu. 5748 Biochemistry 2003, 42, 5748-5753 10.1021/bi034270g CCC: $25.00 © 2003 American Chemical Society Published on Web 04/19/2003