X-ray crystallography reveals a large conformational change during guanyl transfer by mRNA capping enzyme.

AbstractWe have solved the crystal structure of an mRNA capping enzyme at 2.5 Å resolution. The enzyme comprises two domains with a deep, but narrow, cleft between them. The two molecules in the crystallographic asymmetric unit adopt very different conformations; both contain a bound GTP, but one protein molecule is in an open conformation while the other is in a closed conformation. Only in the closed conformation is the enzyme able to bind manganese ions and undergo catalysis within the crystals to yield the covalent guanylated enzyme intermediate. These structures provide direct evidence for a mechanism that involves a significant conformational change in the enzyme during catalysis

An interdomain disulfide bridge links the NtA and first FS domain in agrin

Agrin is a multidomain heparan sulfate proteoglycan involved in postsynaptic differentiation at the neuromuscular junction. Binding of agrin to synaptic basal lamina is mediated by the N-terminal agrin (NtA) domain. The NtA domain of agrin is followed by a tandem of nine follistatin-like (FS) domains forming a rod-like spacer to the laminin G-like domains of the molecule. Here we report that the most C-terminal cysteine residue of NtA (Cys123) forms an interdomain disulfide bond with the FOLN subdomain of the FS module. Remarkably, this single cysteine is flanked by Leu117 and Val124, which are two essential β-branched amino acids forming the heterocomplex of NtA with the γ1 chain of laminin. Moreover, we show that this covalent linkage compensates for the seven amino acid residue splice insert at the very C-terminal helix H3 and causes a rigid interface between NtA and FS independent of the alternative mRNA splice event. These results suggest that the interdomain disulfide bond between the NtA and the first FS domain might be important for the proper folding of agrin