Effects of the N2144S mutation on backbone dynamics of a TB-cbEGF domain pair from human fibrillin-1.
The calcium-binding epidermal growth factor-like (cbEGF) module and the transforming growth factor beta-binding protein-like (TB) module are the two major structural motifs found in fibrillin-1, the extracellular matrix (ECM) protein defective in the Marfan syndrome (MFS). An MFS-causing mutation, N2144S, which removes a calcium ligand in cbEGF32, does not detectably affect fibrillin-1 biosynthesis, rate of secretion, processing, or deposition of reducible fibrillin-1 into the ECM. Since the residue at position 2144 is normally engaged in calcium ligation, it is unable to mediate intermolecular interactions. We have shown previously that this mutation does not affect the folding properties of the TB or cbEGF domains in vitro, but does decrease calcium-binding in cbEGF and TB-cbEGF domain constructs. Here, we use NMR spectroscopy to probe the effects of the N2144S mutation on backbone dynamic properties of TB6-cbEGF32. Analysis of the backbone (15)N relaxation data of wild-type TB6-cbEGF32 has revealed a flexible inter-domain linkage. Parallel dynamics analysis of the N2144S mutant has shown increased flexibility in the region joining the two domains as well as in the calcium-binding site at the N terminus of cbEGF32. This research demonstrates that a small change in peptide backbone flexibility, which does not enhance proteolytic susceptibility of the domain pair, is associated with an MFS phenotype. Flexibility of the TB-cbEGF linkage is likely to contribute to the biomechanical properties of fibrillin-rich connective tissue microfibrils, and may play a role in the microfibril assembly process.
Yuan
Xuemei
X
Department of Biochemistry, University of Oxford, UK.
Werner
Jörn M
JM
Lack
Jeremy
J
Knott
Vroni
V
Handford
Penny A
PA
Campbell
Iain D
ID
Downing
A Kristina
AK
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry; D004058:Diffusion; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000378:metabolism; D000071838:Fibrillin-1; D000071837:Fibrillins; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D053779:Latent TGF-beta Binding Proteins; D009682:Magnetic Resonance Spectroscopy; D008382:Marfan Syndrome / Q000235:genetics; D008840:Microfilament Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009038:Motion; D009154:Mutation / Q000235:genetics*; D018583:Pliability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D054392:Adipokines
D002118:Calcium; D002352:Carrier Proteins; D004815:Epidermal Growth Factor; D000071838:Fibrillin-1; D000071837:Fibrillins; D047908:Intracellular Signaling Peptides and Proteins; D053779:Latent TGF-beta Binding Proteins; D008840:Microfilament Proteins; C000605722:FBN1 protein, human; D054392:Adipokines