title: Crystallization of an engineered RUN domain of Rab6-interacting protein 1/DENND5.
abstract: Effectors of the Rab small GTPases are large multi-domain proteins which have proved difficult to express in soluble form in Escherichia coli. Generally, effectors are recruited to a distinct subcellular compartment by active (GTP-bound) Rabs, which are linked to membranes by one or two prenylated Cys residues at their C-termini. Following recruitment via their Rab-binding domain (RBD), effectors carry out various aspects of vesicle formation, transport, tethering and fusion through their other domains. Previously, successful purification of the RUN-PLAT tandem domains (residues 683-1061) of the 1263-residue Rab6-interacting protein 1 (R6IP1) required co-expression with Rab6, as attempts to solubly express the effector alone were unsuccessful. R6IP1 is also known as DENN domain-containing protein 5 (DENND5) and is expressed as two isoforms, R6IP1A/B (DENND5A/B), which differ by 24 amino acids at the N-terminus. Here, a deletion in R6IP1 was engineered to enable soluble expression and to improve the quality of the crystals grown in complex with Rab6. A large 23-residue loop linking two α-helices in the RUN1 domain was removed and replaced with a short linker. This loop resides on the opposite face to the Rab6-binding site and is not conserved in the RUN-domain family. In contrast to wild-type R6IP1-Rab6 crystals, which took several weeks to grow to full size, the engineered R6IP1 (RPdel)-Rab6 crystals could be grown in a matter of days.
authors.affiliation: School of Biochemistry and Immunology, Trinity College, Dublin 2, Ireland.
authors.forename: Humberto
authors.initials: H
authors.lastname: Fernandes
authors.forename: Edward
authors.initials: E
authors.lastname: Franklin
authors.forename: Amir R
authors.initials: AR
authors.lastname: Khan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003460:Crystallization; D018360:Crystallography, X-Ray; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D015202:Protein Engineering; D016415:Sequence Alignment; D020691:rab GTP-Binding Proteins / Q000737:chemistry*
chemical_list: C094135:Rab6 protein; D020691:rab GTP-Binding Proteins