Yet Another Similarity between Mitochondrial and Bacterial Ribosomal Small Subunit Biogenesis Obtained by Structural Characterization of RbfA from S. aureus.

Ribosome biogenesis is a complex and highly accurate conservative process of ribosomal subunit maturation followed by association. Subunit maturation comprises sequential stages of ribosomal RNA and proteins' folding, modification and binding, with the involvement of numerous RNAses, helicases, GTPases, chaperones, RNA, protein-modifying enzymes, and assembly factors. One such assembly factor involved in bacterial 30S subunit maturation is ribosomal binding factor A (RbfA). In this study, we present the crystal (determined at 2.2 Å resolution) and NMR structures of RbfA as well as the 2.9 Å resolution cryo-EM reconstruction of the 30S-RbfA complex from Staphylococcus aureus (S. aureus). Additionally, we show that the manner of RbfA action on the small ribosomal subunit during its maturation is shared between bacteria and mitochondria. The obtained results clarify the function of RbfA in the 30S maturation process and its role in ribosome functioning in general. Furthermore, given that S. aureus is a serious human pathogen, this study provides an additional prospect to develop antimicrobials targeting bacterial pathogens.

E-site drug specificity of the human pathogen Candida albicans ribosome.

Candida albicans is a widespread commensal fungus with substantial pathogenic potential and steadily increasing resistance to current antifungal drugs. It is known to be resistant to cycloheximide (CHX) that binds to the E-transfer RNA binding site of the ribosome. Because of lack of structural information, it is neither possible to understand the nature of the resistance nor to develop novel inhibitors. To overcome this issue, we determined the structure of the vacant C. albicans 80S ribosome at 2.3 angstroms and its complexes with bound inhibitors at resolutions better than 2.9 angstroms using cryo-electron microscopy. Our structures reveal how a change in a conserved amino acid in ribosomal protein eL42 explains CHX resistance in C. albicans and forms a basis for further antifungal drug development.

A structural view onto disease-linked mutations in the human neutral amino acid exchanger ASCT1.

The ASCT1 transporter of the SLC1 family is largely involved in equilibration of neutral amino acids' pools across the plasma membrane and plays a prominent role in the transport of both L- and D-isomers of serine, essential for the normal functioning of the central nervous system in mammals. A number of mutations in ASCT1 (E256K, G381R, R457W) have been linked to severe neurodevelopmental disorders, however in the absence of ASCT1 structure it is hard to understand their impact on substrate transport. To ameliorate that we have determined a cryo-EM structure of human ASCT1 at 4.2 Å resolution and performed functional transport assays and molecular dynamics simulations, which revealed that given mutations lead to the diminished transport capability of ASCT1 caused by instability of transporter and impeded transport cycle.

Structural and biochemical characterization of a novel ZntB (CmaX) transporter protein from Pseudomonas aeruginosa.

The 2-TM-GxN family of membrane proteins is widespread in prokaryotes and plays an important role in transport of divalent cations. The canonical signature motif, which is also a selectivity filter, has a composition of Gly-Met-Asn. Some members though deviate from this composition, however no data are available as to whether this has any functional implications. Here we report the functional and structural analysis of CmaX protein from a pathogenic Pseudomonas aeruginosa bacterium, which has a Gly-Ile-Asn signature motif. CmaX readily transports Zn2+, Mg2+, Cd2+, Ni2+ and Co2+ ions, but it does not utilize proton-symport as does ZntB from Escherichia coli. Together with the bioinformatics analysis, our data suggest that deviations from the canonical signature motif do not reveal any changes in substrate selectivity or transport and easily alter in course of evolution.

Structural and Functional Characterization of NadR from Lactococcus lactis.

NadR is a bifunctional enzyme that converts nicotinamide riboside (NR) into nicotinamide mononucleotide (NMN), which is then converted into nicotinamide adenine dinucleotide (NAD). Although a crystal structure of the enzyme from the Gram-negative bacterium Haemophilus influenzae is known, structural understanding of its catalytic mechanism remains unclear. Here, we purified the NadR enzyme from Lactococcus lactis and established an assay to determine the combined activity of this bifunctional enzyme. The conversion of NR into NAD showed hyperbolic dependence on the NR concentration, but sigmoidal dependence on the ATP concentration. The apparent cooperativity for ATP may be explained because both reactions catalyzed by the bifunctional enzyme (phosphorylation of NR and adenylation of NMN) require ATP. The conversion of NMN into NAD followed simple Michaelis-Menten kinetics for NMN, but again with the sigmoidal dependence on the ATP concentration. In this case, the apparent cooperativity is unexpected since only a single ATP is used in the NMN adenylyltransferase catalyzed reaction. To determine the possible structural determinants of such cooperativity, we solved the crystal structure of NadR from L. lactis (NadRLl). Co-crystallization with NAD, NR, NMN, ATP, and AMP-PNP revealed a 'sink' for adenine nucleotides in a location between two domains. This sink could be a regulatory site, or it may facilitate the channeling of substrates between the two domains.

Cation permeability in CorA family of proteins.

CorA proteins belong to 2-TM-GxN family of membrane proteins, and play a major role in Mg2+ transport in prokaryotes and eukaryotic mitochondria. The selection of substrate is believed to occur via the signature motif GxN, however there is no consensus how strict this selection within the family. To answer this question, we employed fluorescence-based transport assays on three different family members, namely CorA from bacterium Thermotoga maritima, CorA from the archeon Methanocaldococcus jannaschii and ZntB from bacterium Escherichia coli, reconstituted into proteoliposomes. Our results show that all three proteins readily transport Mg2+, Co2+, Ni2+ and Zn2+, but not Al3+. Despite the similarity in cation specificity, ZntB differs from the CorA proteins, as in the former transport is stimulated by a proton gradient, but in the latter by the membrane potential, confirming the hypothesis that CorA and ZntB proteins diverged to different transport mechanisms within the same protein scaffold.

The structural basis of proton driven zinc transport by ZntB.

Zinc is an essential microelement to sustain all forms of life. However, excess of zinc is toxic, therefore dedicated import, export and storage proteins for tight regulation of the zinc concentration have evolved. In Enterobacteriaceae, several membrane transporters are involved in zinc homeostasis and linked to virulence. ZntB has been proposed to play a role in the export of zinc, but the transport mechanism of ZntB is poorly understood and based only on experimental characterization of its distant homologue CorA magnesium channel. Here, we report the cryo-electron microscopy structure of full-length ZntB from Escherichia coli together with the results of isothermal titration calorimetry, and radio-ligand uptake and fluorescent transport assays on ZntB reconstituted into liposomes. Our results show that ZntB mediates Zn2+ uptake, stimulated by a pH gradient across the membrane, using a transport mechanism that does not resemble the one proposed for homologous CorA channels.