Structural Basis for Ubiquitin Recognition by a Novel Domain from Human Phospholipase A2-activating Protein

Qing-Shan Fu,Chen-Jie Zhou,Hong-Chang Gao,Ya-Jun Jiang,Zi-Ren Zhou,Jing Hong,Wen-Ming Yao,Ai-Xin Song,Dong-Hai Lin,Hong-Yu Hu

doi:10.1074/jbc.m109.009126

Abstract

Ubiquitin (Ub) is an essential modifier conserved in all eukaryotes from yeast to human. Phospholipase A(2)-activating protein (PLAA), a mammalian homolog of yeast DOA1/UFD3, has been proposed to be able to bind with Ub, which plays important roles in endoplasmic reticulum-associated degradation, vesicle formation, and DNA damage response. We have identified a core domain from the PLAA family ubiquitin-binding region of human PLAA (residues 386-465, namely PFUC) that can bind Ub and elucidated its solution structure and Ub-binding mode by NMR approaches. The PFUC domain possesses equal population of two conformers in solution by cis/trans-isomerization, whereas the two isomers exhibit almost equivalent Ub binding abilities. This domain structure takes a novel fold consisting of four beta-strands and two alpha-helices, and the Ub-binding site on PFUC locates in the surface of alpha2-helix, which is to some extent analogous to those of UBA, CUE, and UIM domains. This study provides structural basis and biochemical information for Ub recognition of the novel PFU domain from a PLAA family protein that may connect ubiquitination and degradation in endoplasmic reticulum-associated degradation.

Highlights

Ubiquitin (Ub) is an essential modifier conserved in all eukaryotes from yeast to human
We have identified a core domain from the PLAA family ubiquitin-binding region of human PLAA that can bind Ub and elucidated its solution structure and Ubbinding mode by NMR approaches
The hydrophobic acterized the Ub binding specificity of PLAA by identifying a patch centered by Ile44 may be the major source for the binding novel small domain that is distinct from other known Ub-bind- selectivity of Ub-like proteins or domains with the PLAA family ubiquitin-binding (PFU) domain ing domains in amino acid sequence and three-dimensional of PLAA

Summary

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—The DNA sequences encoding UIM-PFU (residues 312– 465), PFU (residues 340 – 465), and PFUC (residues 386 – 465) were cloned into the pET-32 M vector through BamHI/XhoI sites. All of the mutants of PFUC were generated via PCR and cloned into the pET-32 M vector, which produces Trx-fused proteins. UIM-PFU was cloned into the pGEX-4T-3 vector to produce a GST-fused protein. Chemical Shift Perturbation Experiments—Two-dimensional 1H-15N HSQC spectra of 15N-labeled Ub (ϳ200 ␮M) in an NMR buffer (20 mM phosphate, 50 mM NaCl, 0.01% NaN3, pH 6.5) were recorded at different points of titration with the PLAA fragments, including UIM-PFU, GB1-UIM, PFU, and PFUC and its mutants. 1000 complex structures were generated by rigid body energy minimization, and the best 200 structures with the lowest energy were selected for torsion angle dynamics and Cartesian dynamics calculation in an explicit water solvent It totally produced one major cluster of about 30 structures.

RESULTS

PUF and mutants

The PHD fold of Pru domain from

Findings

DISCUSSION