NMR reveals a different mode of binding of the Stam2 VHS domain to ubiquitin and diubiquitin.
The VHS domain of the Stam2 protein is a ubiquitin binding domain involved in the recognition of ubiquitinated proteins committed to lysosomal degradation. Among all VHS domains, the VHS domain of Stam proteins is the strongest binder to monoubiqiuitin and exhibits preferences for K63-linked chains. In the present paper, we report the solution NMR structure of the Stam2-VHS domain in complex with monoubiquitin by means of chemical shift perturbations, spin relaxation, and paramagnetic relaxation enhancements. We also characterize the interaction of Stam2-VHS with K48- and K63-linked diubiquitin chains and report the first evidence that VHS binds differently to these two chains. Our data reveal that VHS enters the hydrophobic pocket of K48-linked diubiquitin and binds the two ubiquitin subunits with different affinities. In contrast, VHS interacts with K63-linked diubiquitin in a mode similar to its interaction with monoubiquitin. We also suggest possible structural models for both K48- and K63-linked diubiquitin in interaction with VHS. Our results, which demonstrate a different mode of binding of VHS for K48- and K63-linked diubiquitin, may explain the preference of VHS for K63- over K48-linked diubiquitin chains and monoubiquitin.
Université de Lyon, UMR-CNRS 5180 Sciences Analytiques, 69622 Villeurbanne, France.
Anja
A
Lange
Daniela
D
Hoeller
Hans
H
Wienk
Olivier
O
Marcillat
Jean-Marc
JM
Lancelin
Olivier
O
Walker
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D056827:Endosomal Sorting Complexes Required for Transport / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*; D014452:Ubiquitins / Q000737:chemistry / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D056827:Endosomal Sorting Complexes Required for Transport; C491647:STAM2 protein, human; C109184:UBD protein, human; D025801:Ubiquitin; D014452:Ubiquitins