A novel tetrameric PilZ domain structure from xanthomonads.

Tso-Ning Li,Kit-Man Fung,Shan-Ho Chou,Ko-Hsin Chin,Ming-Te Yang,Andrew H-J Wang

doi:10.1371/journal.pone.0022036

Abstract

PilZ domain is one of the key receptors for the newly discovered secondary messenger molecule cyclic di-GMP (c-di-GMP). To date, several monomeric PilZ domain proteins have been identified. Some exhibit strong c-di-GMP binding activity, while others have barely detectable c-di-GMP binding activity and require an accessory protein such as FimX to indirectly respond to the c-di-GMP signal. We now report a novel tetrameric PilZ domain structure of XCC6012 from the plant pathogen Xanthomonas campestris pv. campestris (Xcc). It is one of the four PilZ domain proteins essential for Xcc pathogenicity. Although the monomer adopts a structure similar to those of the PilZ domains with very weak c-di-GMP binding activity, it is nevertheless interrupted in the middle by two extra long helices. Four XCC6012 proteins are thus self-assembled into a tetramer via the extra heptad repeat α3 helices to form a parallel four-stranded coiled-coil, which is further enclosed by two sets of inclined α2 and α4 helices. We further generated a series of XCC6012 variants and measured the unfolding temperatures and oligomeric states in order to investigate the nature of this novel tetramer. Discovery of this new PilZ domain architecture increases the complexity of c-di-GMP-mediated regulation.

Highlights

Recent studies have identified c-di-GMP as a universal secondary messenger molecule that is extensively involved in regulating bacterial pathogenicity [1,2,3,4,5,6,7,8,9]
Due to the highly diverse functions exhibited by c-di-GMP, it is possible that receptors with different or degenerate c-di-GMP binding motifs other than the canonical PilZ domain may exist
XCC6012 contains only a partial c-di-GMP binding signature motif (SxxG, Fig. S2) and a long sequence of 57 amino acids comprising two ahelices (a2 and a3) after the b1 strand (Fig. S2). These results indicate that XCC6012 is a sequence-unique protein from xanthomonads, and is distinctive in the PilZ domain structures

Summary

Introduction

Recent studies have identified c-di-GMP as a universal secondary messenger molecule that is extensively involved in regulating bacterial pathogenicity [1,2,3,4,5,6,7,8,9]. Many PilZ-domain containing proteins from a variety of different bacteria have been found to bind to c-di-GMP with variable affinities, ranging from sub-mM to mM These proteins include PlzD [7] and VCA0042 from V. cholerae [6], YcgR from E. coli and G. xylinus [11], DgrA from C. crescentus [12], and PA4608 [5] and Alg from P. aeruginosa [13]. A Clp (c-AMP receptor protein like protein) from the plant pathogen Xcc was found to be a moderately strong c-di-GMP binder [15,16,17] that is responsible for controlling the expression of approximately 300 downstream genes [18] It lacks the RxxxR and D/NxSxxG motifs present in the canonical PilZ domain [16]. It appears that more alternative c-di-GMP binding motifs will be revealed in further study

Methods

Results

Conclusion