Modeling and analysis of molecularinteraction between Smurf1-WW2 domain and various isoforms of LIM mineralization protein.
LIM Mineralization Protein-1 (LMP-1) has been cloned and shown to be osteoinductive. Our efforts to understand the mode of action of LMP-1 led to the determination that LMP-1 interacts with Smad Ubiquitin Regulatory Factor-1 (Smurf1). Smurf1 targets osteogenic Smads, Smad1/5, for ubiquitin-mediated proteasomal degradation. Smurf1 interaction with LMP-1 or Smads is based on the presence of unique WW-domain interacting motif in these target molecules. By performing site-directed mutagenesis and binding studies in vitro on purified recombinant proteins, we identified a specific motif within the osteogenic region of several LMP isoforms that is necessary for Smurf1 interaction. Similarly, we have identified that the WW2 domain of Smurf1 is necessary for target protein interaction. Here, we present a homology-based modeling of the Smurf1 WW2 domain and its interacting motif of LMP-1. We performed computational docking of the interacting domains in Smurf1 and LMPs to identify the key amino acid residues involved in their binding regions. In support of the computational predictions, we also present biochemical evidence supporting the hypothesis that the physical interaction of Smurf1 and osteoinductive forms of LMP may prevent Smurf1 from targeting osteogenic Smads by ubiquitin-mediated proteasomal degradation.
Sangadala
Sreedhara
S
Atlanta VA Medical Center and the Department of Orthopaedic Surgery, Emory University School of Medicine, Atlanta, Georgia 30329, USA. ssangad@emory.edu
Boden
Scott D
SD
Metpally
Raghu Prasad Rao
RP
Reddy
Boojala Vijay B
BV
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D047908:Intracellular Signaling Peptides and Proteins / Q000302:isolation & purification / Q000378:metabolism*; D060588:LIM Domain Proteins; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D003598:Cytoskeletal Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; D008024:Ligands; C484380:PDLIM7 protein, human; D020033:Protein Isoforms; D025801:Ubiquitin; C498342:SMURF1 protein, human; D044767:Ubiquitin-Protein Ligases