Structural analyses unravel the molecular mechanism of cyclic di-GMP regulation of bacterial chemotaxis via a PilZ adaptor protein

Yan, Xin-Fu; Xin, Lingyi; Yen, Jackie Tan; Zeng, Yukai; Jin, Shengyang; Cheang, Qing Wei; Fong, R.; Chiam, Keng-Hwee; Liang, Zhao‐Xun; Gao, Yong‐Gui

doi:10.1074/jbc.m117.815704

Cited by 21 publications

(23 citation statements)

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“…Structural superposition of PilZ domains from K. xylinus AcsAB, P. aeruginosa Alg44 and C-terminal domains of E. coli YcgR, V. cholerae PlzD, and K. pneumoniae MrkH against the high-resolution structure of the PilZ domain protein MapZ (PA4608) of P. aeruginosa (24, 26, 40) is shown in Fig. 1, while pairwise superpositions of the domains listed in Table 1 are shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Structural conservation and diversity of PilZ-related domains

Galperin

Chou

2019

Preprint

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The widespread bacterial second messenger cyclic diguanylate (c-di-GMP) regulates a 1 variety of processes, including protein secretion, motility, cell development, and biofilm 2 formation. C-di-GMP-dependent responses are often mediated by its binding to the 3 cytoplasmic receptors that contain the PilZ domain. We present here comparative 4 structural and sequence analysis of various PilZ-related domains and describe three 5 principal types of them: (i) the canonical PilZ domain, whose structure includes a six-6 stranded beta-barrel and a C-terminal alpha-helix; (ii) an atypical PilZ domain that 7 contains two extra alpha-helices and forms stable tetramers, and (iii) divergent PilZ-8 related domains, which include the eponymous PilZ protein and PilZN (YcgR_N) and 9 PilZNR (YcgR_2) domains. We refine the second c-di-GMP binding motif of PilZ as 10 [D/N]hSxxG and show that the hydrophobic residue h of this motif interacts with a cluster 11 of conserved hydrophobic residues, helping maintain the PilZ domain fold. We describe 12 several novel PilZN-type domains that are fused to the canonical PilZ domains in specific 13 taxa, such as spirochetes, actinobacteria, cellulosolytic clostridia, deltaproteobacteria, 14 and aquificae. We propose that the evolution of three major groups of PilZ domains 15 included (i) fusion of pilZ with other genes, which produced Alg44, cellulose synthase, and 16 other multidomain proteins; (ii) insertion of a ~200-bp fragment, which resulted in the 17 formation of tetramer-forming PilZ proteins, and (iii) tandem duplication of pilZ genes, 18 which led to the formation of PilZ dimers and YcgR-like proteins. 19 IMPORTANCE 20Cyclic di-GMP is a ubiquitous bacterial second messenger that regulates motility, biofilm 21 formation and virulence of many bacterial pathogens. The PilZ domain is a widespread c-22 di-GMP receptor that binds c-di-GMP through its RxxxR and [D/N]xSxxG motifs; some PilZ 23 domains lack these motifs and are unable to bind c-di-GMP. We used structural and 24 sequence analysis to assess the diversity of PilZ-related domains and define their common 25 features. We show that the hydrophobic residue h in the second motif is highly conserved; 26 it may serve as a readout for c-di-GMP binding. We describe three principal classes of 27 PilZ-related domains: canonical, tetramer-forming, and divergent PilZ domains, and 28 propose the evolutionary pathways that led to the emergence of these PilZ types. 29 30

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Section: Resultsmentioning

confidence: 99%

Structural conservation and diversity of PilZ-related domains

Galperin

Chou

2019

Preprint

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“…We classified this group of proteins as singledomain PilZ proteins considering that they only contain a core PilZ domain, an N-terminal loop and a short C-terminal motif. The variation in protein length in the single-domain PilZ proteins is mainly due to the differences in the C-terminal region, with secondary structure analysis 7 suggesting that the C-terminal region exhibits great structural diversity by adopting helical, strand or coil secondary structure. The second-largest group of PilZ proteins (9, 053 in total) contains 140 -280 residues; and we classified them as di-domain PilZ proteins because they possess an additional C-or N-terminal protein domain.…”

Section: Classification Of Pilz Proteins Into Single Di-and Multi-domentioning

confidence: 99%

“…In vitro and in vivo studies revealed that MapZ binds c-di-GMP with a KD in the range of 6-9 µM 5, 32 , and that it interacts with the chemotaxis methyltransferase CheR1 at elevated c-di-GMP concentrations. Crystallographic studies on the MapZ/CheR1/c-di-GMP ternary complex identified that the two C-terminal helices α1 (residues 92-106aa) and α2 (residues 109-121) of MapZ are the key motifs mediating MapZ-CheR1 interaction ( Figure 6C) 7,33 . In particular, the helix α1 plays an essential role in mediating the MapZ-CheR1 interaction by occupying the central cleft of CheR1.…”

Section: A Large Group Of Single-domain Pilz Proteins Predicted To Inmentioning

confidence: 99%

“…A large number of di-domain PilZ proteins are predicted to be YcgR-like proteins that feature an N-terminal YcgRN-like domain and a C-terminal c-di-GM-binding PilZ domain. In the SSN, YcgR-like proteins constitute 38% of the total proteins and many are found in large clusters (Clusters 1,4,5,6,7,8,9 and 13). PilZ domain is also fused to a variety of other non-YcgRN domains to form non-YcgR-type di-domain proteins.…”

Section: Overview Of the Ssn For Di-domain Pilz Proteinsmentioning

confidence: 99%

“…Canonical PilZ domains bind monomeric or dimeric c-di-GMP with a wide range of affinities using two key motifs: the arginine-rich motif (RxxxR) from the N-terminal loop and the (D/N)xSxxG motif from the β-strands [4][5][6][7] . Apart from the canonical PilZ domains, noncanonical PilZ domain proteins that lack c-di-GMP-binding capability have also been discovered [8][9][10][11] .…”

Section: Introductionmentioning

confidence: 99%

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Large-scale sequence similarity analysis reveals the scope of sequence and function divergence in PilZ domain proteins

Cheang

Sheng

et al. 2020

Preprint

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PilZ domain-containing proteins constitute a superfamily of widely distributed bacterial signalling proteins. Although studies have established the canonical PilZ domain as an adaptor protein domain evolved to specifically bind the second messenger c-di-GMP, mounting evidence suggest that the PilZ domain has undergone enormous divergent evolution to generate a superfamily of proteins that are characterized by a wide range of c-di-GMP-binding affinity, binding partners and cellular functions. The divergent evolution has even generated families of non-canonical PilZ domains that completely lack c-di-GMP binding ability. In this study, we performed a large-scale sequence analysis on more than 28,000 single-and di-domain PilZ proteins using the sequence similarity networking tool created originally to analyse functionally diverse enzyme superfamilies. The sequence similarity networks (SSN) generated by the analysis feature a large number of putative isofunctional protein clusters, and thus, provide an unprecedented panoramic view of the sequence-function relationship and function diversification in PilZ proteins. Some of the protein clusters in the networks are considered as unexplored clusters that contain proteins with completely unknown biological function;whereas others contain one, two or a few functionally known proteins, and therefore, enabling us to infer the cellular function of uncharacterized homologs or orthologs. With the ultimate goal of elucidating the diverse roles played by PilZ proteins in bacterial signal transduction, the work described here will facilitate the annotation of the vast number of PilZ proteins encoded by bacterial genome and help to prioritize functionally unknown PilZ proteins for future studies.3 Importance Although PilZ domain is best known as the protein domain evolved specifically for the binding of the second messenger c-di-GMP, divergent evolution has generated a superfamily of PilZ proteins with a diversity of ligand or protein-binding properties and cellular functions.We analysed the sequences of more than 28,000 PilZ proteins using the sequence similarity networking (SSN) tool to yield a global view of the sequence-function relationship and function diversification in PilZ proteins. The results will facilitate the annotation of the vast number of PilZ proteins encoded by bacterial genomes and help us prioritize PilZ proteins for future studies.

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Structural analyses of the AAA+ ATPase domain of the transcriptional regulator GtrR in the BDSF quorum‐sensing system in Burkholderia cenocepacia

et al. 2021

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Global transcriptional regulator downstream RpfR (GtrR) is a key downstream regulator for quorum-sensing signaling molecule cis-2-dodecenoic acid (BDSF). As a bacterial enhancer-binding protein (bEBP), GtrR is composed of an N-terminal receiver domain, a central ATPases associated with diverse cellular activities (AAA+) ATPase r 54 -interaction domain, and a C-terminal helix-turn-helix DNA-binding domain. In this work, we solved its AAA+ ATPase domain in both apo and GTP-bound forms. The structure revealed how GtrR specifically recognizes GTP. In addition, we also revealed that GtrR has moderate GTPase activity in vitro in the absence of its activation signal. Finally, we found the residues K170, D236, R311, and R357 in GtrR that are crucial to its biological function, any single mutation leading to completely abolishing GtrR activity.

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Structural analyses unravel the molecular mechanism of cyclic di-GMP regulation of bacterial chemotaxis via a PilZ adaptor protein

Cited by 21 publications

References 40 publications

Structural conservation and diversity of PilZ-related domains

Structural conservation and diversity of PilZ-related domains

Large-scale sequence similarity analysis reveals the scope of sequence and function divergence in PilZ domain proteins

Structural analyses of the AAA+ ATPase domain of the transcriptional regulator GtrR in the BDSF quorum‐sensing system in Burkholderia cenocepacia

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