Binding of Cyclic Diguanylate in the Non-catalytic EAL Domain of FimX Induces a Long-range Conformational Change

Abstract

FimX is a multidomain signaling protein required for type IV pilus biogenesis and twitching motility in the opportunistic pathogen Pseudomonas aeruginosa. FimX is localized to the single pole of the bacterial cell, and the unipolar localization is crucial for the correct assembly of type IV pili. FimX contains a non-catalytic EAL domain that lacks cyclic diguanylate (c-di-GMP) phosphodiesterase activity. It was shown that deletion of the EAL domain or mutation of the signature EVL motif affects the unipolar localization of FimX. However, it was not understood how the C-terminal EAL domain could influence protein localization considering that the localization sequence resides in the remote N-terminal region of the protein. Using hydrogen/deuterium exchange-coupled mass spectrometry, we found that the binding of c-di-GMP to the EAL domain triggers a long-range (ϳca. 70 Å ) conformational change in the N-terminal REC domain and the adjacent linker. In conjunction with the observation that mutation of the EVL motif of the EAL domain abolishes the binding of c-di-GMP, the hydrogen/deuterium exchange results provide a molecular explanation for the mediation of protein localization and type IV pilus biogenesis by c-di-GMP through a remarkable allosteric regulation mechanism.

Cyclic di-GMP: the First 25 Years of a Universal Bacterial Second Messenger

“…These "retired from active duty" domains have evolved to carry out new functions. One of these functions may involve binding (but not processing) of the substrate, e.g., GTP binding in the A sites of inactive GGDEF domains (44) or c-di-GMP binding in the substrate binding sites of enzymatically inactive EAL domains (85,101,133). Another set of functions of GGDEF, EAL, and HD-GYP domains that have "retired" from catalysis includes their participation in protein-protein or protein-RNA interactions.…”

“…An interesting example of such a hybrid protein is P. aeruginosa FimX, involved in type IV pilus-based motility. The degenerate and enzymatically inactive C-terminal EAL domain of FimX serves as a high-affinity c-di-GMP receptor (85,101,133). Another example involves the GGDEF-EAL c-di-GMP receptor LapD from Pseudomonas fluorescens (166,167).…”

“…Type IV pilus biogenesis, but not gene expression, requires the GGDEF-EAL domain FimX response regulator localized at one cell pole (100). FimX senses c-di-GMP via its degenerate EAL domain (85,133). Although the molecular mechanisms by which FimX affects type IV pilus biogenesis and twitching motility are unknown for P. aeruginosa, studies of the FimX homolog in X. axonopodis suggest that FimX interacts with a PilZ protein (Fig.…”

Global Regulatory Pathways and Cross-talk ControlPseudomonas aeruginosaEnvironmental Lifestyle and Virulence Phenotype

“…Consistent with this hypothesis, mutation of the GGDEF domain in FimX, abolishes GTP binding and stimulation of PDE activity (Kazmierczak, 2006). However, recent structural studies indicate that the degenerate GGDEF domain of FimX is incapable of nucleotide binding, while the EAL domain binds c-di-GMP with high affinity inducing a conformational change that may impede FimX binding to its putative partner located at the bacterial pole to regulate Tfp production and twitching motility (Navarro, 2010;Qi, 2011).…”

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

“…In contrast, many nucleotide-binding proteins were captured with a relatively low specificity and, to a large degree, are probably false positives. Further validation of the specific binding to c-di-GMP using techniques such as DRaCALA 20 , UV cross-linking 15 , differential scanning fluorimetry (DSF) 21 , Microscale thermophoresis (MST) 22 , isothermal calorimetry (ITC) [23][24] … is thus necessary.…”