Catherine Joseph

Three−dimensional structure and stability of the KH domain: molecular insights into the fragile X syndrome

The KH module is a sequence motif found in a number of proteins that are known to be in close association with RNA. Experimental evidence suggests a direct involvement of KH in RNA binding. The human FMR1 protein, which has two KH domains, is associated with fragile X syndrome, the most common inherited cause of mental retardation. Here we present the three-dimensional solution structure of the KH module. The domain consists of a stable beta alpha alpha beta beta alpha fold. On the basis of our results, we suggest a potential surface for RNA binding centered on the loop between the first two helices. Substitution of a well-conserved hydrophobic residue located on the second helix destroys the KH fold; a mutation of this position in FMR1 leads to an aggravated fragile X phenotype.

Ca2+-independent binding of an EF-hand domain to a novel motif in the alpha-actinin-titin complex.

The interaction between α-actinin and titin, two modular muscle proteins, is essential for sarcomere assembly. We have solved the solution structure of a complex between the calcium-insensitive C-terminal EF-hand domain of α-actinin-2 and the seventh Z-repeat of titin. The structure of the complex is in a semi-open conformation and closely resembles that of myosin light chains in their complexes with heavy chain IQ motifs. However, no IQ motif is present in the Z-repeat, suggesting that the semi-open conformation is a general structural solution for calcium-independent recognition of EF-hand domains.

Secondary structure determination by NMR spectroscopy of an immunoglobulin-like domain from the giant muscle protein titin

SummaryWe present the complete 15N and 1H NMR assignment and the secondary structure of an immunoglobulin-like domain from the giant muscle protein titin. The assignment was obtained using homonuclear and 15N heteronuclear 2D and 3D experiments. The complementarity of 3D TOCSY-NOESY and 3D 15N NOESY-HSQC experiments, using WATERGATE for water suppression, allowed an efficient assignment of otherwise ambiguous cross peaks and was helpful in overcoming poor TOCSY transfer for some amino acids. The secondary structure is derived from specific NOEs between backbone α- and amide protons, secondary chemical shifts of α-protons and chemical exchange for the backbone amide protons. It consists of eight β-strands, forming two β-sheets with four strands each, similar to the classical β-sandwich of the immunoglobulin superfamily, as previously predicted by sequence analysis. Two of the β-strands are connected by type II β-turns; the first β-strand forms a β-bulge. The whole topology is very similar to the only intracellular immunoglobulin-like domain for which a structure has been determined so far, i.e., telokin.