The aspartate aminotransferase-like domain of Firmicutes MocR transcriptional regulators
Graphical abstract
Introduction
The GntR family of bacterial transcriptional regulators has been recently described (Haydon and Guest, 1991) and named after the repressor of the gluconate operon in Bacillus subtilis. GntR regulators possess an N-terminal domain containing a conserved helix-turn-helix (HTH) module able to recognize specific DNA motifs and a C-terminal effector-binding and/or oligomerization domain (Eb/O). According to current models, binding of the effector molecule to the Eb/O domain would induce a conformational change that influences the DNA-binding properties of the HTH domain. Within the GntR family, the structure of the C-terminus domain is heterogeneous and, depending on its nature, several subfamilies can be identified (Hoskisson and Rigali, 2009). The MocR subfamily is particularly interesting because it is distinguished by a domain homologous to fold type-I pyridoxal 5′-phosphate (PLP) enzymes (Paiardini et al., 2004, Percudani and Peracchi, 2009, Schneider et al., 2000). The type-I archetypal protein is aspartate aminotransferase (AAT), the first PLP-dependent enzyme of fold type-I whose three-dimensional structure has been elucidated (Kirsch et al., 1984). AAT-like PLP-enzymes were until recently deemed to exist mainly as stand-alone homodimers or as higher order complexes. It is now emerging, after scrutiny of genomic sequence data from prokaryotes, that fold type-I proteins can be found also in multidomain contexts such as nonribosomal peptide synthetases (Milano et al., 2013). The MocR regulators themselves represent a multidomain framework hosting AAT-like modules.
MocR regulators are widespread among eubacteria genomes, although with heterogeneity (Bramucci et al., 2011, Hoskisson and Rigali, 2009): apparently, they are rare or even absent in bacteria phyla characterized by compact genomes (size of about 1.0–2.0 Mb) such as Chlamydiae and Mollicutes, while in other species are present with a variable number of paralogous genes. For example, one MocR-like protein has been found only in the Mollicutes bacterium HR1 (genome size 1.88 Mb) out of 52 Mollicutes complete proteomes tested. Likewise, a single MocR regulator has been found in the bacterium Opitutus terrae (genome size 5.9 Mb) out of 20 Chlamydiae complete proteomes scrutinized (Bramucci et al., 2011). Only a few members of the MocR family have been structurally and functionally characterized: for example, GabR that regulates the utilization of γ-aminobutyric acid (GABA) in B. subtilis (Belitsky, 2004) and PdxR from Listeria monocytogenes and Corynebacterium glutamicum, involved in the regulation of PLP biosynthesis (Belitsky, 2014, Jochmann et al., 2011). Recently, the crystallographic structure of the GabR protein, deposited in the Protein Data Bank (PDB) with the codes 4MGR, 4N0B, and 4TV7 (Edayathumangalam et al., 2013, Okuda et al., 2015), confirmed the presence of a fold type-I domain at the C-terminus of the regulator and opened new perspectives in deciphering the mechanism of action of MocRs. GabR is a domain swap homodimer in which the HTH domain of one subunit interacts with the PLP-binding domain of the other subunit; this is possible thanks to a very long linker that connects the two domains.
Currently, a comprehensive structural analysis of the MocR family is missing. The aim of this work was to give a contribution to the understanding of the structural characteristics of the AAT-like domain of these regulators, and to shed light on its structural and functional relationship with PLP-dependent enzymes. For ease of data analysis, our work has been restricted to the Firmicutes phylum. Our results also provide a rational guide for site-directed mutagenesis studies aimed at deciphering the structure–function relationships in these new and intriguing family of regulators.
Section snippets
Data collection
Complete Firmicutes proteomes were retrieved from the UniProt database release 2013–11 (UniProt, 2014). Putative MocR sequences were identified using the software HMMER 3.0 (Finn et al., 2011) while the required seed multiple alignment was calculated with the program MUSCLE version 3.8 (Bramucci et al., 2011, Edgar, 2004). Data analysis was assisted by in-house written Perl, Python and Bash scripts. The retrieved putative MocR sequences were compared to the models of the Pfam (Protein families)
Sequence and phylogenetic analyses
The alignment of the 117 AAT-like domains selected from the initial set of 699 sequences is displayed in Supplementary Fig. 1. The corresponding tree is reported in Fig. 1 while the “nucleotidic” tree is displayed in Supplementary Fig. 2.
The phylogenetic noise of the nucleotidic and amino acidic data set was investigated by means of likelihood mapping. The percentage of dots falling in the central area of the triangles was about 11% for both nucleotidic and aminoacidic data sets, confirming
Discussion
The AAT-like domains of Firmicutes MocRs are rather divergent except for very specific areas of functional relevance: the “active” site, the subunit interface and small portions of the surface possibly involved in DNA or protein interactions. This observation suggests that emergence of the ancestor of MocR subfamily was a primordial event.
Genes coding for MocR regulators are widespread in bacterial genomes and often present in multiple paralogous copies (Bramucci et al., 2011, Hoskisson and
Acknowledgments
This research has been partially funded by a grant from Sapienza, University of Rome, for the year 2013. TM was supported by a grant (“assegno di ricerca”) from Sapienza University of Rome and Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”. Authors thank Prof. Sebastiana Angelaccio for critically reading the manuscript.
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