A cationic lumen in the Wzx flippase mediates anionic O-antigen subunit translocation in Pseudomonas aeruginosa PAO1.

Heteropolymeric B-band O-antigen (O-Ag) biosynthesis in Pseudomonas aeruginosa PAO1 follows the Wzy-dependent pathway, beginning with translocation of undecaprenyl pyrophosphate-linked anionic O-Ag subunits (O units) from the inner to the outer leaflets of the inner membrane (IM). This translocation is mediated by the integral IM flippase Wzx. Through experimentally based and unbiased topological mapping, our group previously observed that Wzx possesses many charged and aromatic amino acid residues within its 12 transmembrane segments (TMS). Herein, site-directed mutagenesis targeting 102 residues was carried out on the TMS and loops of Wzx, followed by assessment of each construct's ability to restore B-band O-Ag production, identifying eight residues important for flippase function. The importance of various charged and aromatic residues was highlighted, predominantly within the TMS of the protein, revealing functional 'hotspots' within the flippase, particularly within TMS2 and TMS8. Construction of a tertiary structure homology model for Wzx indicated that TMS2 and TMS8 line a central cationic lumen. This is the first report to describe a charged flippase lumen for mediating anionic O-unit translocation across the hydrophobic IM.

ProQ is an RNA chaperone that controls ProP levels in Escherichia coli.

Transporter ProP mediates osmolyte accumulation in Escherichia coli cells exposed to high osmolality media. The cytoplasmic ProQ protein amplifies ProP activity by an unknown mechanism. The N- and C-terminal domains of ProQ are predicted to be structurally similar to known RNA chaperone proteins FinO and Hfq from E. coli. Here we demonstrate that ProQ is an RNA chaperone, binding RNA and facilitating both RNA strand exchange and RNA duplexing. Experiments performed with the isolated ProQ domains showed that the FinO-like domain serves as a high-affinity RNA-binding domain, whereas the Hfq-like domain is largely responsible for RNA strand exchange and duplexing. These data suggest that ProQ may regulate ProP production. Transcription of proP proceeds from RpoD- and RpoS-dependent promoters. Lesions at proQ affected ProP levels in an osmolality- and growth phase-dependent manner, decreasing ProP levels when proP was expressed from its own chromosomal promoters or from a heterologous plasmid-based promoter. Small RNA molecules are known to regulate cellular levels of sigma factor RpoS. ProQ did not act by changing RpoS levels since proQ lesions did not influence RpoS-dependent stationary phase thermotolerance and they affected ProP production and activity similarly in bacteria without and with an rpoS defect. Taken together, these results suggest that ProQ does not regulate proP transcription. It may act as an RNA-binding protein to regulate proP translation.

Transmembrane helix I and periplasmic loop 1 of Escherichia coli ProP are involved in osmosensing and osmoprotectant transport.

Osmoregulatory transporters stimulate bacterial growth by mediating osmoprotectant uptake in response to increasing osmotic pressure. The ProP protein of Escherichia coli transports proline and other osmoprotectants. Like LacY, ProP is a member of the major facilitator superfamily and a H(+)-solute symporter. ProP is regulated by osmotic pressure via a membrane potential-dependent mechanism. A homology model predicts that ionizable and polar residues, highly conserved among ProP homologues, cluster deep within the N-terminal helix bundle of ProP. Chemical labeling of introduced cysteine (Cys) residues supported the homology model by confirming the predicted positions of transmembrane helix I (TMI) and periplasmic loop 1. Replacements of residues in the putative polar cluster impaired or altered ProP function, suggesting that they are important for osmosensing and may interact with the transport substrates. Asn34, Glu37, Phe41, Tyr44, and Ala48 line the most polar face of TMI; Tyr44 is on the periplasmic side of the putative polar cluster, and Ala59 is in periplasmic loop 1. The N-ethylmaleimide reactivities of Cys introduced at positions 41, 44, 48, and 59 increased with osmotic pressure, whereas the reactivities of those at cytoplasm-proximal positions 34 and 37 did not. Replacements of polar cluster residues that blocked transport also affected the NEM reactivity of Cys44 and its osmolality dependence. This report and previous work suggest that conformational changes associated with osmosensing may shift the equilibria between outward- and inward-facing transport pathway intermediates.

Structural modeling identified the tRNA-binding domain of Utp8p, an essential nucleolar component of the nuclear tRNA export machinery of Saccharomyces cerevisiae.

Utp8p is an essential 80 kDa intranuclear tRNA chaperone that transports tRNAs from the nucleolus to the nuclear tRNA export receptors in Saccharomyces cerevisiae. To help understand the mechanism of Utp8p function, predictive tools were used to derive a partial model of the tertiary structure of Utp8p. Secondary structure prediction, supported by circular dichroism measurements, indicated that Utp8p is divided into 2 domains: the N-terminal beta sheet and the C-terminal alpha helical domain. Tertiary structure prediction was more challenging, because the amino acid sequence of Utp8p is not directly homologous to any known protein structure. The tertiary structures predicted by threading and fold recognition had generally modest scores, but for the C-terminal domain, threading and fold recognition consistently pointed to an alpha-alpha superhelix. Because of the sequence diversity of this fold type, no single structural template was an ideal fit to the Utp8p sequence. Instead, a composite template was constructed from 3 different alpha-alpha superhelix structures that gave the best matches to different portions of the C-terminal domain sequence. In the resulting model, the most conserved sequences grouped in a tight cluster of positive charges on a protein that is otherwise predominantly negative, suggesting that the positive-charge cleft may be the tRNA-binding site. Mutations of conserved positive residues in the proposed binding site resulted in a reduction in the affinity of Utp8p for tRNA both in vivo and in vitro. Models were also derived for the 10 fungal homologues of Utp8p, and the localization of the positive charges on the conserved surface was found in all cases. Taken together, these data suggest that the positive-charge cleft of the C-terminal domain of Utp8p is involved in tRNA-binding.

Periplasmic loops of osmosensory transporter ProP in Escherichia coli are sensitive to osmolality.

ProP is an osmosensory transporter. The activities of ProP and ProP*, a cysteine-less, His(6)-tagged ProP variant, increase with osmotic pressure in cells and proteoliposomes. In proteoliposomes, ProP activity is osmolality-dependent only if the magnitude of the membrane potential (DeltaPsi) exceeds 100 mV. Some amino acid replacements rendered ProP activity osmolality-insensitive [e.g., Y44M in transmembrane segment 1 (TMI); S62C in periplasmic loop 1 (loop P1)], whereas others elevated the osmolality at which ProP activates (e.g., A59C). This suggested that the environments and/or conformations of TMI and loop P1 might be osmolality-dependent. This report correlates structural dynamics of ProP with osmoregulation of its transport activity. Residues in periplasmic loops were replaced with Cys, and changes in their environments were detected by monitoring their reactivities with N-ethylmaleimide (NEM). Increasing osmolality markedly increased the NEM reactivity of some Cys residues (e.g., C59, loop P1; C415-C418, loop P6) but not others (e.g., C293, loop P4; C348, loop P5). The NEM reactivity of C62 was insensitive to osmolality, as expected. Substitution Y44M rendered the transport activities of ProP*-A59C and ProP*-Q415C, and the NEM reactivities of the introduced Cys, osmolality-insensitive. Furthermore, osmolality did not affect the reactivity of C59 in cells lacking a protonmotive force, consistent with evidence that DeltaPsi is required for osmosensing by ProP. These results indicate that the osmotically induced increases in NEM reactivity of C59 and C415 in energized bacteria are due to a conformational change of ProP in response to osmolality. They therefore constitute the first direct evidence of an osmotically induced conformational change associated with osmosensing by a transporter.

Core residue replacements cause coiled-coil orientation switching in vitro and in vivo: structure-function correlations for osmosensory transporter ProP.

Protein ProP acts as an osmosensory transporter in diverse bacteria. C-Terminal residues 468-497 of Escherichia coli ProP (ProPEc) form a four-heptad homodimeric alpha-helical coiled coil. Arg 488, at a core heptad a position, causes it to assume an antiparallel orientation. Arg in the hydrophobic core of coiled coils is destabilizing, but Arg 488 forms stabilizing interstrand salt bridges with Asp 475 and Asp 478. Mutation R488I destabilizes the coiled coil and elevates the osmotic pressure at which ProPEc activates. It may switch the coiled-coil orientation to parallel by eliminating the salt bridges and increasing the hydrophobicity of the core. In this study, mutations D475A and D478A, which disrupt the salt bridges without increasing the hydrophobicity of the coiled-coil core, had the expected modest impacts on the osmotic activation of ProPEc. The five-heptad coiled coil of Agrobacterium tumefaciens ProP (ProPAt) has K498 and R505 at a positions. Mutation K498I had little effect on the osmotic activation of ProPAt, and ProPAt-R505I was activated only at high osmotic pressure; on the other hand, the double mutant was refractory to osmotic activation. Both a synthetic peptide corresponding to ProPAt residues 478-516 and its K498I variant maintained the antiparallel orientation. The single R505I substitution created an unstable coiled coil with little orientation preference. Double mutation K498I/R505I switched the alignment, creating a stable parallel coiled coil. In vivo cross-linking showed that the C-termini of ProPAt and ProPAt-K498I/R505I were antiparallel and parallel, respectively. Thus, the antiparallel orientation of the ProP coiled coil is contingent on Arg in the hydrophobic core and interchain salt bridges. Two key amino acid replacements can convert it to a stable parallel structure, in vitro and in vivo. An intermolecular antiparallel coiled coil, present on only some orthologues, lowers the osmotic pressure required to activate ProP. Formation of a parallel coiled coil renders ProP inactive.

The structure and function of Saccharomyces cerevisiae proteinase A.

Saccharomyces cerevisiae proteinase A (saccharopepsin; EC 3.4.23.25) is a member of the aspartic proteinase superfamily (InterPro IPR001969), which are proteolytic enzymes distributed among a variety of organisms. Targeted to the vacuole as a zymogen, its activation at acidic pH can occur by two different pathways, a one-step process to release mature proteinase A, involving the intervention of proteinase B, or a step-wise pathway via the autoactivation product known as pseudo-proteinase A. Once active, S. cerevisiae proteinase A is essential to the activities of other yeast vacuolar hydrolases, including proteinase B and carboxypeptidase Y. The mature enzyme is bilobal, with each lobe providing one of the two catalytically essential aspartic acid residues in the active site. The crystal structure of free proteinase A reveals that the flap loop assumes an atypical position, pointing directly into the S(1) pocket of the enzyme. With regard to hydrolysis, proteinase A has a preference for hydrophobic residues with Phe, Leu or Glu at the P1 position and Phe, Ile, Leu or Ala at P1', and is inhibited by IA(3), a natural and highly specific inhibitor produced by S. cerevisiae. This review is the first comprehensive review of S. cerevisiae PrA.

Structure and function of transmembrane segment XII in osmosensor and osmoprotectant transporter ProP of Escherichia coli.

Escherichia coli transporter ProP acts as both an osmosensor and an osmoregulator. As medium osmolality rises, ProP is activated and mediates H+-coupled uptake of osmolytes like proline. A homology model of ProP with 12-transmembrane (TM) helices and cytoplasmic termini was created, and the protein's topology was substantiated experimentally. Residues 468-497, at the end of the C-terminal domain and linked to TM XII, form an intermolecular, homodimeric alpha-helical coiled-coil that tunes the transporter's response to osmolality. We aim to further define the structure and function of ProP residues Q415-E440, predicted to include TM XII. Each residue was replaced with cysteine (Cys) in a histidine-tagged, Cys-less ProP variant (ProP*). Cys at positions 415-418 and 438-440 were most reactive with Oregon Green Maleimide (OGM), suggesting that residues 419 through 437 are in the membrane. Except for V429-I433, reactivity of those Cys varied with helical periodicity. Cys predicted to face the interior of ProP were more reactive than Cys predicted to face the lipid. The former may be exposed to hydrated polar residues in the protein interior, particularly on the periplasmic side. Intermolecular cross-links formed when ProP* variants with Cys at positions 419, 420, 422, and 439 were treated with DTME. Thus TM XII can participate, along its entire length, in the dimer interface of ProP. Cys substitution E440C rendered ProP* inactive. All other variants retained more than 30% of the proline uptake activity of ProP* at high osmolality. Most variants with Cys substitutions in the periplasmic half of TM XII activated at lower osmolalities than ProP*. Variants with Cys substitutions on one face of the cytoplasmic half of TM XII required a higher osmolality to activate. They included elements of a GXXXG motif that are predicted to form the interface of TM XII with TM VII. These studies define the position of ProP TM XII within the membrane, further support the predicted structure of ProP, reveal the dimerization interface, and show that the structure of TM XII influences the osmolality at which ProP activates.

Evidence that WbpD is an N-acetyltransferase belonging to the hexapeptide acyltransferase superfamily and an important protein for O-antigen biosynthesis in Pseudomonas aeruginosa PAO1.

Di-N-acetylated uronic acid residues are unique sugar moieties observed in the lipopolysaccharides (LPS) of respiratory pathogens including several serotypes of Pseudomonas aeruginosa and several species of Bordetella. WbpD of P. aeruginosa PAO1 (serotype O5) is a putative 3-N-acetyltransferase that has been implicated in the biosynthesis of UDP-2,3-diacetamido-2,3-dideoxy-d-mannuronic acid [UDP-d-Man(2NAc3NAc)A], a precursor for the d-Man(2NAc3NAc)A residues in the B-band O antigen of this bacterium. A chromosomal knockout mutant of wbpD is incapable of producing either long-chain B-band O antigen (> or = 2 repeating units) or semi-rough LPS (lipid A-core + one repeat). Adding wbpD in trans restored LPS production to the wild-type level; this indicates that wbpD is important for biosynthesis of individual B-band O-antigen repeating units. WbpD contains left-handed beta-helical (LbetaH) structure as observed by Conserved Domain analysis and in silico secondary and tertiary structure predictions. This feature suggested that WbpD belongs to the hexapeptide acyltransferase (HexAT) superfamily of enzymes. WbpD was overexpressed as an N-terminally histidine-tagged fusion protein (His6-WbpD) and purified to > 95% purity. The protein was subjected to Far-UV circular dichroism spectroscopy, and the data revealed that WbpD contains left-handed helical structure, which substantiated in silico predictions made earlier. Results from SDS-PAGE, matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS), and gel filtration analyses indicated that His6-WbpD has trimeric organization, consistent with the quaternary structure of HexATs. The binding of acetyl-CoA by WbpD was demonstrated by MALDI-TOF MS, suggesting that WbpD is an acetyltransferase that utilizes a direct-transfer reaction mechanism. Incubation of WbpD with acetyl-CoA significantly enhanced the stability of the protein and prevented precipitation over a course of 14 days. As a substrate for studying the enzymatic activity of WbpD is unavailable at present, a structure-based model for the LbetaH domain of WbpD was generated. Comparisons between this model and the LbetaH domains of known HexATs suggested that Lys136 plays a role in acetyl-CoA binding. A K136A site-directed mutant construct could only partially complement the wbpD knockout, and this mutation also reduced the stabilizing effects of acetyl-CoA, while a K136R mutation showed no discernible effect on complementation of the wbpD mutant or the stabilizing effects of acetyl-CoA on the purified mutant protein. A modified pathway was proposed for the biosynthesis of UDP-d-Man(2NAc3NAc)A, in which WbpD is involved in the catalysis of the fourth step by acting as a UDP-2-acetamido-3-amino-2,3-dideoxy-d-glucuronic acid 3-N-acetyltransferase.

A structural model for the osmosensor, transporter, and osmoregulator ProP of Escherichia coli.

Transporter ProP of Escherichia coli, a member of the major facilitator superfamily (MFS), acts as an osmosensor and an osmoregulator in cells and after purification and reconstitution in proteoliposomes. H(+)-osmoprotectant symport via ProP is activated when medium osmolality is elevated with membrane impermeant osmolytes. The three-dimensional structure of ProP was modeled with the crystal structure of MFS member GlpT as a template. This GlpT structure represents the inward (or cytoplasm)-facing conformation predicted by the alternating access model for transport. LacZ-PhoA fusion analysis and site-directed fluorescence labeling substantiated the membrane topology and orientation predicted by this model and most hydropathy analyses. The model predicts the presence of a proton pathway within the N-terminal six-helix bundle of ProP (as opposed to the corresponding pathway found within the C-terminal helix bundle of its paralogue, LacY). Replacement of residues within the N-terminal helix bundle impaired the osmotic activation of ProP, providing the first indication that residues outside the C-terminal domain are involved in osmosensing. Some residues that were accessible from the periplasmic side, as predicted by the structural model, were more susceptible to covalent labeling in permeabilized membrane fractions than in intact bacteria. These residues may be accessible from the cytoplasmic side in structures not represented by our current model, or their limited exposure in vivo may reflect constraints on transporter structure that are related to its osmosensory mechanism.

Overexpression, purification, and characterization of ProQ, a posttranslational regulator for osmoregulatory transporter ProP of Escherichia coli.

ProP is an osmosensor and osmoregulatory transporter in Escherichia coli. Osmotic activation of ProP is attenuated 5-fold in the absence of soluble protein ProQ, but proQ lesions do not influence proP transcription or ProP levels. The mechanism by which ProQ amplifies ProP activity is unknown. Putative proQ orthologues are found in Gram-negative bacteria (only), but none have known functions. ProQ was overexpressed to low and high levels with and without a C-terminal histidine tag (His(6)). Plasmid-encoded ProQ or ProQ-His(6) complemented in-frame chromosomal deletion DeltaproQ676, restoring ProP activity. After overexpression, both proteins were poorly soluble unless cells were lysed in media of high salinity. ProQ copurified with DNA binding proteins of similar size (HU and a histone-like protein) by ion exchange and exclusion chromatographies, whereas ProQ-His(6) could be purified to homogeneity by nickel chelate affinity chromatography. Sequence-based analysis and modeling suggest that ProQ includes distinct N- and C-terminal domains linked by an unstructured sequence. The N-terminal domain can be modeled on the crystal structure of alpha-helical RNA binding protein FinO, whereas the C-terminal domain can be modeled on an SH3-like domain (beta-structure). Both ProQ and ProQ-His(6) appeared to be monomeric, though the higher Stokes radius of ProQ-His(6) may reflect altered domain interactions. The measured secondary structure content of ProQ (circular dichroism (CD) spectroscopy) contrasted with sequence-based prediction but was as expected if the spectrum of the C-terminal domain is analogous to those reported for SH3 domains. The CD spectrum of ProQ was pH- but not NaCl-sensitive.

The Pseudomonas aeruginosa initiation factor IF-2 is responsible for formylation-independent protein initiation in P. aeruginosa.

Formylation of the initiator methionyl-tRNA (Met-tRNAfMet) was generally thought to be essential for initiation of protein synthesis in all eubacteria based on studies conducted primarily in Escherichia coli. However, this view of eubacterial protein initiation has changed because some bacteria have been demonstrated to have the capacity to initiate protein synthesis with the unformylated Met-tRNAfMet. Here we show that the Pseudomonas aeruginosa initiation factor IF-2 is required for formylation-independent protein initiation in P. aeruginosa, the first bacterium shown to have the ability to initiate protein synthesis with both the initiator formyl-methionyl-tRNA (fMet-tRNAfMet) and Met-tRNAfMet. The E. coli IF-2, which participates exclusively in formylation-dependent protein initiation in E. coli, was unable to facilitate utilization of Met-tRNAfMet in initiation in P. aeruginosa. However, the E. coli IF-2 was made to function in formylation-independent protein initiation in P. aeruginosa by decreasing the positive charge potential of the cleft that binds the amino end of the amino acid attached to the tRNA. Furthermore increasing the positive charge potential of this cleft in the P. aeruginosa IF-2 prevented the protein from participating in formylation-independent protein initiation. Thus, this is the first demonstration of a eubacterial IF-2 with an inherent capacity to facilitate utilization of Met-tRNAfMet in protein initiation, discounting the dogma that eubacterial IF-2 can only allow the use of fMet-tRNAfMet in protein initiation. Furthermore these findings give important clues to the basis for discriminating the initiator Met-tRNA by IF-2 and for the evolution of alternative mechanisms for discrimination.