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J. Biol. Chem., Vol. 282, Issue 3, 2091-2100, January 19, 2007

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Direct Demonstration of the Flexibility of the Glycosylated Proline-Threonine Linker in the Cellulomonas fimi Xylanase Cex through NMR Spectroscopic Analysis^*

David K. Y. Poon^¶, Stephen G. Withers^¶, and Lawrence P. McIntosh^¶||1

From the Department of Biochemistry and Molecular Biology, Department of Chemistry, ^¶The Protein Engineering Network of Centres of Excellence, and ^||The Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada

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 (47 kDa) and glycosylated (51 kDa) forms. The PT linker lacks any predominant structure in either form as indicated by random coil amide chemical shifts. Furthermore, heteronuclear ¹H-¹⁵N 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 ¹⁵N 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 cellulosebinding 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.

Received for publication, October 13, 2006 , and in revised form, November 20, 2006.

^* This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada with instrumentation support provided by the Protein Engineering Network of Centres of Excellence, the Canadian Foundation for Innovation, the British Columbia Knowledge Development Fund, the University of British Columbia Blusson Fund, the Michael Smith Foundation for Health Research, and the Canadian Institutes of Health Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Life Sciences Centre, 2350 Health Sciences Mall, University of British Columbia, Vancouver, BC V6T 1Z3, Canada. Tel.: 604-822-3341; Fax: 604-822-5227; E-mail: mcintosh{at}chem.ubc.ca.

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