Structural studies of the Enterococcus faecalis SufU [Fe-S] cluster protein

Abstract

Background: Iron-sulfur clusters are ubiquitous and evolutionarily ancient inorganic prosthetic groups, the biosynthesis of which depends on complex protein machineries. Three distinct assembly systems involved in the maturation of cellular Fe-S proteins have been determined, designated the NIF, ISC and SUF systems. Although well described in several organisms, these machineries are poorly understood in Gram-positive bacteria. Within the Firmicutes phylum, the Enterococcus spp. genus have recently assumed importance in clinical microbiology being considered as emerging pathogens for humans, wherein Enterococcus faecalis represents the major species associated with nosocomial infections. The aim of this study was to carry out a phylogenetic analysis in Enterococcus faecalis V583 and a structural and conformational characterisation of it SufU protein.

Results: BLAST searches of the Enterococcus genome revealed a series of genes with sequence similarity to the Escherichia coli SUF machinery of [Fe-S] cluster biosynthesis, namely sufB, sufC, sufD and SufS. In addition, the E. coli IscU ortholog SufU was found to be the scaffold protein of Enterococcus spp., containing all features considered essential for its biological activity, including conserved amino acid residues involved in substrate and/or co-factor binding (Cys50,76,138 and Asp52) and, phylogenetic analyses showed a close relationship with orthologues from other Gram-positive bacteria. Molecular dynamics for structural determinations and molecular modeling using E. faecalis SufU primary sequence protein over the PDB:1su0 crystallographic model from Streptococcus pyogenes were carried out with a subsequent 50 ns molecular dynamic trajectory. This presented a stable model, showing secondary structure modifications near the active site and conserved cysteine residues. Molecular modeling using Haemophilus influenzae IscU primary sequence over the PDB:1su0 crystal followed by a MD trajectory was performed to analyse differences in the C-terminus region of Gram-positive SufU and Gram-negative orthologous proteins, in which several modifications in secondary structure were observed.

Conclusion: The data describe the identification of the SUF machinery for [Fe-S] cluster biosynthesis present in the Firmicutes genome, showing conserved sufB, sufC, sufD and sufS genes and the presence of the sufU gene coding for scaffold protein, instead of sufA; neither sufE nor sufR are present. Primary sequences and structural analysis of the SufU protein demonstrated its structural-like pattern to the scaffold protein IscU nearby on the ISC machinery. E. faecalis SufU molecular modeling showed high flexibility over the active site regions, and demonstrated the existence of a specific region in Firmicutes denoting the Gram positive region (GPR), suggested as a possible candidate for interaction with other factors and/or regulators.

Figure 1

The biosynthetic machinery for [Fe-S] cluster formation in Gram-positive bacteria. (A) Comparison of the genetic organization of genes involved in the [Fe-S] cluster assembly. Genes having homologous sequences or similar functions between the two systems are color-coded: E. coli ISC and SUF machineries and conserved ORFs coding for putative SUF machinery in Gram-positive bacteria. (B) Neighbour-Joining phylogenetic analysis of protein sequences. (C) Comparison of sequences from members of the NifU/IscU/SufU orthologues. Cysteines are presented as yellow, aspartate as green, LPPVK of IscU in red, and the characteristic Gram-positive insertion in blue.

Figure 2

Representation of crystallographic and MD conformations of SufU. (A) Template S. pyogenes SufU crystal (PDB ID 1SU0); (B) SufU S. pyogenes conformation after 50 ns; (C) SufU E. faecalis model conformation after 50 ns. SufU structural characteristics are presented (α-helices I-IV and β-sheet a-c), as well as the characteristic Gram-positive region of 19 amino acids (GPR). (D) Active showing different conformations of active sites residues before and after MD trajectory: Cys residues are shaded in yellow and orange, and Asp in green and cyan blue for crystal and MD conformations, respectively.

Figure 3

Root Mean Square Deviation (RMSD) analysis. Root Mean Square Deviation (RMSD) for the entire protein (A), N-terminal (B) and C-terminal (C) regions, and (D) radius of gyration (template SufU is presented in black and E. faecalis SufU in red).

Figure 4

Flexibility analysis. Sausage plot for (A) template S. pyogenes SufU and (D) model E. faecalis SufU proteins. The thickness presented is directly related to the flexibility in the area. Loops are presented in gray, α-helices in red and β-strands in blue. Root Mean Square Fluctuation (RMSF) analysis, as a function of both residue number and time for (B) template S. pyogenes and (C) E. faecalis SufU proteins, presenting the four α-helices (H), the three β-strands (S), and the GPR region.