The folding and aggregation properties of a single KH-domain protein: Ribosome binding factor A (RbfA) from Pseudomonas aeruginosa
Introduction
The hnRNP K homology (KH) domains are small domains consisting of around 70 amino acids, present in different proteins in archaea, bacteria and eukaryotes [1]. Typically, KH domains are found in multiple copies functioning either independently or cooperatively, however few examples of proteins with single KH motifs have been found [1]. These domains are characterized by the ability to bind RNA or ssDNA and are often located in proteins involved in different steps of RNA metabolism, regulation of gene expression [2,3] and in ribosome biogenesis [4]. In humans, the loss of function of specific KH domains have been reported to play a role in several diseases including fragile X mental retardation syndrome and cancer [1].
From a structural point of view, KH domains consist of three α-helices packed onto the surface of a central three-stranded β sheet; depending on the position of two additional α and β elements with respect to the β1α1α2β2 core, they are classified into two distinct topologies: the eukaryotic Type I KH-domain (KHI), with additional α and β elements at the C-terminus, and the prokaryotic Type II KH-domain (KHII), with additional α and β elements at the N-terminus [1,3]. Recently, the existence of a third KH topology fold (KHIII) has been proposed [5]. Most KH domains, albeit not all, contain a conserved sequence motif (the GxxG loop) located between helices α1 and α2, which has been proposed to be essential for nucleic acid binding [4].
Ribosome-binding factor A (RbfA) is a small cold-shock ribosome assembly factor [6] composed by a single KH domain, that assists the maturation of the 30S subunit [[7], [8], [9]]. At low temperature, the expression level of RbfA increases rapidly [10] allowing the bacterial cells to effectively overcome the translational blockage occurring at these extreme conditions [7,8]. On the basis of structural and biochemical studies it has been proposed that RbfA cooperates with RsgA, another ribosome assembly factor, to allow the proper folding of the 30S subunit at the late stage of the small subunit maturation [[11], [12], [13]]. Recently it has been speculated that the human orthologue of bacterial RbfA plays a key function in the quality control of mitochondrial ribosome assembly [14].
The available structures of RbfA proteins from different bacterial species, such as Haemophilus influenzae (HiRbfA; PDB ID: 1JOS, unpublished), Escherichia coli (EcRbfA; PDB ID: 1KKG) [6], Thermotoga maritima (TmRbfA; PDB ID: 2KZF, unpublished), Thermus thermophilus (TtRbfA; PDB ID: 2DYJ) [8] and Mycoplasma pneumoniae (MpRbfA; PDB ID:1PA4) [15], show that they all belong to the KHII domain sub-class.
The role played by the KH domains in various strategic cellular functions and their involvement in some pathologies, prompted recent outcomes that highlight some crucial aspects of their structure, function and nucleic acid recognition ability [1,3,14]. However, although the ability of proteins to fold, misfold or aggregate is of paramount importance for their cellular functions, to our knowledge, limited information is available on these dynamic properties in the case of KH domains.
To fill this gap, here we report a biophysical characterization of RbfA from the pathogenic bacterium Pseudomonas aeruginosa (PaRbfA). PaRbfA is a small protein (129 aminoacids) composed by a single structured KH domain followed by a C-terminal region and represents a useful system to unveil both the aggregation properties and the folding mechanism of this bacterial protein domain. Our results show that, in vitro, PaRbfA can form ordered fibrils endowed with cross-β structure even in mild conditions, a remarkable feature that could be of general interest for other proteins containing KH-domain(s). Moreover, fluorescence-based experiments, carried out on a fluorescent pseudo-wild-type engineered variant of the protein (PaRbfA-Y77W), allowed us to determine the thermodynamic stability of PaRbfA and to propose a mechanism of folding which highlighted the presence of a transiently populated folding intermediate.
Section snippets
Equilibrium denaturation of PaRbfA
PaRbfA was expressed in E. coli and purified to homogeneity (yield ≈ 30 mg/L) as reported in Materials and Methods. The secondary structure content of recombinant PaRbfA is consistent with a α/β protein such as the KH domains, as judged from its far-UV CD spectra obtained at temperatures below 50 °C (Fig. 1A). Interestingly, the far-UV CD spectra obtained at temperatures above 50 °C show a broad negative band centered at ≈ 215 nm, atypical for a denatured polypeptide chain and suggesting a
Discussion
Despite the central role of KH domain in several patho-physiological processes, both in prokaryotes and eukaryotes [1,3], little is known about the biophysical properties and folding dynamics of this domain. In this context, folding studies can contribute to characterize the (mis)folding properties of the KH domains, to identify the presence of metastable intermediate states (if any), to depict their structural properties and unveil their role in the folding mechanism. PaRbfA offers a unique
Cloning, mutagenesis, expression, and purification
The rbfA coding sequence was PCR amplified from the genomic DNA of P. aeruginosa PAO1, using primers rbfA_FW (5’-ggaattccatATGGCAAAAGACTACAGCCG-3′) and rbfA_RV (5’-ccgctcgaGCTTCACCTGGGCCACGC-3′), digested with NdeI and XhoI, and cloned into the pET28(b) + vector (Novagen) previously digested with the same enzymes. The construct encoding the Y77W variant was obtained using the QuickChange Lightning Site-Directed Mutagenesis kit (Agilent technologies) according to the manufacturer's instructions,
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was partially supported by "Sapienza" University of Rome Grant 2017 n° RM11715C7F529A09 to CTA.
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