Structure of a SUMO-binding-motif mimic bound to Smt3p-Ubc9p: conservation of a non-covalent ubiquitin-like protein-E2 complex as a platform for selective interactions within a SUMO pathway.
The SUMO ubiquitin-like proteins play regulatory roles in cell division, transcription, DNA repair, and protein subcellular localization. Paralleling other ubiquitin-like proteins, SUMO proteins are proteolytically processed to maturity, conjugated to targets by E1-E2-E3 cascades, and subsequently recognized by specific downstream effectors containing a SUMO-binding motif (SBM). SUMO and its E2 from the budding yeast Saccharomyces cerevisiae, Smt3p and Ubc9p, are encoded by essential genes. Here we describe the 1.9 A resolution crystal structure of a non-covalent Smt3p-Ubc9p complex. Unexpectedly, a heterologous portion of the crystallized complex derived from the expression construct mimics an SBM, and binds Smt3p in a manner resembling SBM binding to human SUMO family members. In the complex, Smt3p binds a surface distal from Ubc9's catalytic cysteine. The structure implies that a single molecule of Smt3p cannot bind concurrently to both the non-covalent binding site and the catalytic cysteine of a single Ubc9p molecule. However, formation of higher-order complexes can occur, where a single Smt3p covalently linked to one Ubc9p's catalytic cysteine also binds non-covalently to another molecule of Ubc9p. Comparison with other structures from the SUMO pathway suggests that formation of the non-covalent Smt3p-Ubc9p complex occurs mutually exclusively with many other Smt3p and Ubc9p interactions in the conjugation cascade. By contrast, high-resolution insights into how Smt3p-Ubc9p can also interact with downstream recognition machineries come from contacts with the SBM mimic. Interestingly, the overall architecture of the Smt3p-Ubc9p complex is strikingly similar to recent structures from the ubiquitin pathway. The results imply that non-covalent ubiquitin-like protein-E2 complexes are conserved platforms, which function as parts of larger assemblies involved in many protein post-translational regulatory pathways.
Duda
David M
DM
Department of Structural Biology and Genetics, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
van Waardenburg
Robert C A M
RC
Borg
Laura A
LA
McGarity
Sierra
S
Nourse
Amanda
A
Waddell
M Brett
MB
Bjornsti
Mary-Ann
MA
Schulman
Brenda A
BA
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D006801:Humans; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D012097:Repressor Proteins / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D025841:Small Ubiquitin-Related Modifier Proteins; D025801:Ubiquitin / Q000737:chemistry*; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry*; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D011506:Proteins; D012097:Repressor Proteins; C099362:SMT3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D025801:Ubiquitin; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9; D003545:Cysteine