|Title||Direct demonstration of the flexibility of the glycosylated proline-threonine linker in the Cellulomonas fimi xylanase Cex through NMR spectroscopic analysis|
|Publication Type||Journal Article|
|Year of Publication||2007|
|Authors||Poon, DKY, Withers, SG, McIntosh, LP|
|Journal||Journal of Biological Chemistry|
The modular xylanase Cex ( or CfXyn10A) from Cellulomonas fimi consists of an N-terminal catalytic domain and a C-terminal cellulose- binding domain, joined by a glycosylated proline-threonine ( PT) linker. To characterize the conformation and dynamics of the Cex linker and the consequences of its modification, we have used NMR spectroscopy to study full-length Cex in its nonglycosylated ( similar to 47 kDa) and glycosylated ( similar to 51 kDa) forms. The PT linker lacks any predominant structure in either form as indicated by random coil amide chemical shifts. Furthermore, heteronuclear H-1-N-15 nuclear Overhauser effect relaxation measurements demonstrate that the linker is flexible on the ns-to-ps time scale and that glycosylation partially dampens this flexibility. The catalytic and cellulose- binding domains also exhibit identical amide chemical shifts whether in isolation or in the context of either unmodified or glycosylated full-length Cex. Therefore, there are no noncovalent interactions between the two domains of Cex or between either domain and the linker. This conclusion is supported by the distinct N-15 relaxation properties of the two domains, as well as their differential alignment within a magnetic field by Pf1 phage particles. These data demonstrate that the PT linker is a flexible tether, joining the structurally independent catalytic and cellulose-binding domains of Cex in an ensemble of conformations; however, more extended forms may predominate because of restrictions imparted by the alternating proline residues. This supports the postulate that the binding-domain anchors Cex to the surface of cellulose, whereas the linker provides flexibility for the catalytic domain to hydrolyze nearby hemicellulose ( xylan) chains.
|URL||<Go to ISI>://000243451300060|