Structure analysis of the global metabolic regulator Crc from Pseudomonas aeruginosa

Abstract

The global metabolic regulator catabolite repression control (Crc) has recently been found to modulate the susceptibility to antibiotics and virulence in the opportunistic pathogen Pseudomonas aeruginosa and been suggested as a nonlethal target for novel antimicrobials. In P. aeruginosa, Crc couples with the CA motifs from the small RNA CrcZ to form a posttranscriptional regulator system and is removed from the 5 0 -end of the target mRNAs. In this study, we first reported the crystal structure of Crc from P. aeruginosa refined to 2.20 Å. The structure showed that it consists of two halves with similar overall topology and there are 11 b strands surrounded by 13 helices, forming a four-layered a/b-sandwich. The circular dichroism spectroscopy revealed that it is thermostable in solution and shares similar characteristics to that in crystal.Comprehensive structural analysis and comparison with the homologies of Crc showed high similarity with several known nucleases and consequently may be classified into a member exodeoxyribonuclease III. However, it shows distinct substrate specificity (RNA as the preferred substrate) compared to these DNA endonucleases. Structural comparisons also revealed potential RNA recognition and binding region mainly consisting of five flexible loops. Our structure study provided the basis for the future application of Crc as a target to develop new antibiotics. V C 2012 IUBMB Life, 65(1): [50][51][52][53][54][55][56][57] 2013

The Pseudomonas aeruginosa Catabolite Repression Control Protein Crc Is Devoid of RNA Binding Activity

“…Here, we report the crystal structure of the P. aeruginosa Crc refined to 1.8 Å. At variance with a recent report [19] our structural analyses did not corroborate intrinsic nucleic acid binding properties of Crc. Using different DNA substrates, we show that Crc is devoid of DNA nuclease activity, which can be reconciled with an alteration of the catalytically active site when compared to the AP endonuclease family.…”

“…Although the amino acid sequence identities between Crc and these proteins are only 25 to 37% (Figure S1), the superposition of Crc with the available structure of the AP endonuclease hApe1 (PDB accession code 1BIX, Figure 1A) revealed high structural similarity with a root mean square deviation (RMSD) of 1.59 Å over 253 superposed Cα atoms. The comparison of the Crc structure obtained in this study with a recently published structure of Crc (PDB accession code 4F1R) resolved at 2.20 Å resolution [19] revealed only minimal differences with a RMSD of 0.26 Å over 259 superposed Cα atoms.…”

Architectural principles for Hfq/Crc-mediated regulation of gene expression

“…The assembly has C2 symmetry, with the molecular twofold axis passing through the interface of the two Crc molecules. The Crc´s in the assembly self-interact in the same way as observed in the crystal structure of the isolated Crc dimer, where the interface is generated through crystallographic symmetry (Wei et al, 2013; Milojevic et al, 2013). As anticipated from mutational analyses (Sonnleitner et al, 2018), the dominating protein/RNA interaction is made by the distal face of Hfq, forming an interface area of roughly 2270 Å 2 .…”