A G1127S change in calcium-binding epidermal growth factor-like domain 13 of human fibrillin-1 causes short range conformational effects

Human fibrillin-1, an extracellular matrix glycoprotein, has a modular orga nization that includes 43 calcium-binding epidermal growth factor-like (cbE GF) domains arranged as multiple tandem repeats. A missense mutation that c hanges a highly conserved glycine to serine (G1127S) has been identified in cbEGF13, which results in a variant of Marfan syndrome, a connective tissu e disease. Previous experiments on isolated cbEGF13 and a cbEGF13-14 pair i ndicated that the G1127S mutation caused defective folding of cbEGF13 but n ot cbEGF14, We have used limited proteolysis methods and two-dimensional NM R spectroscopy to identify the structural consequences of this mutation in a covalently linked cbEGF12-13 pair and a cbEGF12-14 triple domain construc t. Protease digestion studies of the cbEGF12-13 G1127S mutant pair indicate d that both cbEGF12 and 13 retained similar calcium binding properties and thus tertiary structure to the normal domain pair, because all identified c leavage sites showed calcium-dependent protection from proteolysis. However , small changes in the conformation of cbEGF13 G1127S, revealed by the pres ence of a new protease-sensitive site and comparative two-dimensional NOESY data, suggested that the fold of the mutant domain was not identical to th e wild-type, but was native-like. Additional cleavage sites identified in c bEGF12-14 G1127S indicated further subtle changes within the mutant domain but not the flanking domains, We have concluded the following in this study . (i) Covalent linkage of cbEGF12 preserves the native-like fold of cbEGF13 G1127S and (ii) conformational effects introduced by G1127S are localized to cbEGF13, This study demonstrates that missense mutations in fibrillin-1 cbEGF domains can cause short range structural effects in addition to long range effects previously observed with a E1073K mutation in cbEGF12.