The accessory gas vesicle protein GvpM of haloarchaea and its interaction partners during gas vesicle formation

Gas vesicles consist predominantly of the hydrophobic GvpA and GvpC, and the accessory proteins GvpF through GvpM are required in minor amounts during formation. GvpM and its putative interaction partners were investigated. GvpM interacted with GvpH, GvpJ and GvpL, but not with GvpG. Interactions were also observed in vivo in Haloferax volcanii transformants using Gvp fusions to the green fluorescent protein smGFP. Cells producing the hydrophobic M_GFP contained a single fluorescent aggregate per cell, whereas cells containing L_GFP or H_GFP were fully fluorescent. The soluble L_GFP formed stable co-aggregates with GvpM in L_GFPM transformants, but the presence of GvpH resulted in the absence of M_GFP foci in HM_GFP transformants. Substitution- and deletion mutants of GvpM determined functionally important amino acids (aa). Substitution of a polar by a non-polar aa in the N-terminal region of GvpM had no effect, whereas a substitution of a non-polar by a polar aa in this region inhibited gas vesicle formation in transformants. Substitutions in region 44–48 of GvpM strongly reduced the number of gas vesicles, and deletions at the N-terminus resulted in Vac^? transformants. Gas vesicle morphology was not affected by any mutation, implying that GvpM is required during initial stages of gas vesicle assembly.     

The homologies of gas vesicle proteins.

In addition to GvpA, the main structural protein, an SDS-soluble protein has been found in gas vesicles isolated from six different genera of cyanobacteria. N-terminal sequence analysis of the first 30 to 60 residues of the gel-purified proteins showed that they were homologous to GvpC, a protein that strengthens the gas vesicle in Anabaena flos-aquae. The proteins from some of the organisms showed rather low homology, however, and this may explain why the genes that encode them have not been found by Southern hybridization studies. The gas vesicles of another cyanobacterium, Dactylococcopsis salina, contained two SDS-soluble proteins (M(r) 17,000 and 35,000) that were identical in sequence for the first 24 residues but not thereafter; these two proteins showed no clear homology to GvpC. The sequence of GvpA, the main structural gas vesicle protein, was very similar in each of the organisms investigated. GvpA from the purple bacterium Amoebobacter pendens was different for the first 8 residues but 51 of the next 56 residues were identical to those of the cyanobacterial GvpA. Analysis of the GvpA and GvpC sequences provides support for the idea that the low diversity of GvpA reflects a high degree of conservation rather than a recent origin followed by lateral gene transfer between different bacteria.     

Functional analysis of the gas vesicle gene cluster of the halophilic archaeon Haloferax mediterranei defines the vac-region boundary and suggests a regulatory role for the gvpD gene or its product

A series of deletions introduced into the gvp gene cluster of Haloferax mediterranei, comprising 14 genes involved in gas vesicle synthesis (mc-vac-region), was investigated by transformation experiments. Gas vesicle production and the expression of the gvpA gene encoding the major gas vesicle protein, GvpA, was monitored in each Haloferax volcanii transformant. Whereas transformants containing the entire mc-vac-region produced gas vesicles (Vac+), various deletions in the region 5' to gvpA (encompassing gvpD-gvpM) or 3' to gvpA (containing gvpC, gvpN and gvpO) revealed Vac- transformants. All these transformants expressed gvpA and contained the 8 kDa GvpA protein as shown by Western analysis. However, transformants containing the gvpA gene by itself indicated a lower level of GvpA than observed with each of the other transformants. None of these transformants containing deletion constructs assembled the GvpA protein into gas vesicles. In contrast, transformants containing a construct carrying a 918 bp deletion internal to gvpD exhibited a tremendous gas vesicle overproduction, suggesting a regulatory role for the gvpD gene or its product. This is the first assignment of a functional role for one of the 13 halobacterial gvp genes found in addition to gvpA that are involved in the synthesis of this unique structure.     

Structural model of the gas vesicle protein GvpA and analysis of GvpA mutants in vivo

Gas vesicles are gas-filled protein structures increasing the buoyancy of cells. The gas vesicle envelope is mainly constituted by the 8 kDa protein GvpA forming a wall with a water excluding inner surface. A structure of GvpA is not available; recent solid-state NMR results suggest a coil-α-β-β-α-coil fold. We obtained a first structural model of GvpA by high-performance de novo modelling. Attenuated total reflection (ATR)-Fourier transform infrared spectroscopy (FTIR) supported this structure. A dimer of GvpA was derived that could explain the formation of the protein monolayer in the gas vesicle wall. The hydrophobic inner surface is mainly constituted by anti-parallel β-strands. The proposed structure allows the pinpointing of contact sites that were mutated and tested for the ability to form gas vesicles in haloarchaea. Mutations in α-helix I and α-helix II, but also in the β-turn affected the gas vesicle formation, whereas other alterations had no effect. All mutants supported the structural features deduced from the model. The proposed GvpA dimers allow the formation of a monolayer protein wall, also consistent with protease treatments of isolated gas vesicles.     

Solution structure of protein synthesis initiation factor 1 from Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic bacterial pathogen and a primary cause of nosocomial infection in humans. The rate of antibiotic resistance in P. aeruginosa is increasing worldwide leading to an unmet need for discovery of new chemical compounds distinctly different from present antimicrobials. Protein synthesis is an essential metabolic process and a validated target for the development of new antibiotics. Initiation factor 1 from P. aeruginosa (Pa‐IF1) is the smallest of the three initiation factors that act to establish the 30S initiation complex during initiation of protein biosynthesis. Here we report the characterization and solution NMR structure of Pa‐IF1. Pa‐IF1 consists of a five‐stranded β‐sheet with an unusual extended β‐strand at the C‐terminus and one short α‐helix arranged in the sequential order β1‐β2‐β3‐α1‐β4‐β5. The structure adopts a typical β‐barrel fold and contains an oligomer‐binding motif. A cluster of basic residues (K39, R41, K42, K64, R66, R70, and R72) located on the surface of strands β4 and β5 near the short α‐helix may compose the binding interface with the 30S subunit.     

Modeling of the major gas vesicle protein, GvpA: from protein sequence to vesicle wall structure

The structure and assembly process of gas vesicles have received significant attention in recent decades, although relatively little is still known. This work combines state-of-the-art computational methods to develop a model for the major gas vesicle protein, GvpA, and explore its structure within the assembled vesicle. Elucidating this protein's structure has been challenging due to its adherent and aggregative nature, which has so far precluded in-depth biochemical analyses. Moreover, GvpA has extremely low similarity with any known protein structure, which renders homology modeling methods ineffective. Thus, alternate approaches were used to model its tertiary structure. Starting with the sequence from haloarchaeon Halobacterium sp. NRC-1, we performed ab initio modeling and threading to acquire a multitude of structure decoys, which were equilibrated and ranked using molecular dynamics and mechanics, respectively. The highest ranked predictions exhibited an α-β-β-α secondary structure in agreement with earlier experimental findings, as well as with our own secondary structure predictions. Afterwards, GvpA subunits were docked in a quasi-periodic arrangement to investigate the assembly of the vesicle wall and to conduct simulations of contact-mode atomic force microscopy imaging, which allowed us to reconcile the structure predictions with the available experimental data. Finally, the GvpA structure for two representative organisms, Anabaena flos-aquae and Calothrix sp. PCC 7601, was also predicted, which reproduced the major features of our GvpA model, supporting the expectation that homologous GvpA sequences synthesized by different organisms should exhibit similar structures.     

Complexity of gas vesicle biogenesis in Halobacterium sp. strain NRC-1: identification of five new proteins

The genome of Halobacterium sp. strain NRC-1 contains a large gene cluster, gvpMLKJIHGFEDACNO, that is both necessary and sufficient for the production of buoyant gas-filled vesicles. Due to the resistance of gas vesicles to solubilization, only the major gas vesicle protein GvpA and a single minor protein, GvpC, were previously detected. Here, we used immunoblotting analysis to probe for the presence of gas vesicle proteins corresponding to five additional gvp gene products. Polyclonal antisera were raised in rabbits against LacZ-GvpF, -GvpJ, and -GvpM fusion proteins and against synthetic 15-amino-acid peptides from GvpG and -L. Immunoblotting analysis was performed on cell lysates of wild-type Halobacterium sp. strain NRC-1, gas vesicle-deficient mutants, and purified gas vesicles, after purification of LacZ fusion antibodies on protein A and beta-galactosidase affinity columns. Our results show the presence of five new gas vesicle proteins (GvpF, GvpG, GvpJ, GvpL, and GvpM), bringing the total number of proteins identified in the organelles to seven. Two of the new gas vesicle proteins are similar to GvpA (GvpJ and GvpM), and two proteins contain predicted coiled-coil domains (GvpF and GvpL). GvpL exhibited a multiplet ladder on sodium dodecyl sulfate-polyacrylamide gels indicative of oligomerization and self-assembly. We discuss the possible functions of the newly discovered gas vesicle proteins in biogenesis of these unique prokaryotic flotation organelles.     

Complementation studies with the gas vesicle-encoding p-vac region of Halobacterium salinarium PHH1 reveal a regulatory role for the p-gvpDE genes

Gas-vesicle (Vac) synthesis in Halobacterium salinarium PHH1 involves the expression of the p-vac region consisting of 14 different gvp genes that are arranged in two clusters: p-gvpACNO and, oppositely oriented, p-gvpDEFGHIJKLM. The latter cluster of genes is transcribed as two units: p-gvpDE and p-gvpF–M. The 5′-terminus of the p-gvpF–M mRMA was located 169 nucleotides upstream of p-gvpF within p-gvpE. The p-gvpG and p-gvpK gene was expressed in Escherichia coli and antibodies to proteins obtained were raised in rabbits. Both proteins could be detected in halobacterial cell lysates; in gas-vesicle preparations, however, neither GvpG nor GvpK could be found. The requirement for single p-gvp gene expression for gas-vesicle synthesis was determined by transformation experiments using the Vac^? species Haloferax volcanii as recipient. Construct ΔA containing all p-gvp genes except for p-gvpA, encoding the major gas-vesicle structural protein, produced Vac^? transformants, but the addition of p-gvpA on a second vector restored gas-vesicle synthesis to wild-type level (Vac⁺⁺). Similarly, double transformants containing p-gvpD–M plus p-gvpACNO, or p-gvpG–M (fused to the promoter of the halobacterial ferredoxin gene for expression) plus p-gvpFED–ACNO were Vac⁺⁺. Transformants containing the p-vac region either lacking gvpA, gvpF, or gvpGHI were Vac^?, indicating the absolute requirement of these gvp genes (or at least one in the case of gvpGHI) for gas-vesicle formation. Double transformants containing the constructs p-gvpF–M plus p-gvpACNO (ΔDE) accumulated gas vesicles (Vac⁺) but synthesized fewer than the wild type, showing that the p-gvpDE genes are not necessary for gas-vesicle assembly. A repressor function affecting the synthesis of the p-gvpF–M mRNA could be suggested for p-gvpD and the 5′- region of its mRNA.     

The sequence of the major gas vesicle protein,GvpA, influences the width and strength of halobacterial gas vesicles

Transformation experiments with Haloferax volcanii show that the amino acid sequence of the gas vesicle protein GvpA influences the morphology and strength of gas vesicles produced by halophilic archaea. A modified expression vector containing p-gvpA was used to complement a Vac(-) strain of Hfx. volcanii that harboured the entire p-vac region (from Halobacterium salinarum PHH1) except for p-gvpA. Replacement of p-gvpA with mc-gvpA (from Haloferax mediterranei) led to the synthesis of gas vesicles that were narrower and stronger. Other gene replacements (using c-gvpA from Hbt. salinarum or mutated p-gvpA sequences) led to a significant but smaller increase in gas vesicle strength, and less marked effects on gas vesicle morphology.     

Involvement of β‐adrenergic receptor in synaptic vesicle swelling and implication in neurotransmitter release

Secretory vesicle swelling is required for vesicular discharge during cell secretion. The G_αo‐mediated water channel aquaporin‐6 (AQP‐6) involvement in synaptic vesicle (SV) swelling in neurons has previously been reported. Studies demonstrate that in the presence of guanosine triphosphate (GTP), mastoparan, an amphiphilic tetradecapeptide from wasp venom, activates G_o protein GTPase, and stimulates SV swelling. Stimulation of G proteins is believed to occur via insertion of mastoparan into the phospholipid membrane to form a highly structured α‐helix that resembles the intracellular loops of G protein‐coupled adrenergic receptors. Consequently, the presence of adrenoceptors and the presence of an endogenous β‐adrenergic agonist at the SV membrane is suggested. Immunoblot analysis of SV using β‐adrenergic receptor antibody, and vesicle swelling experiments using β‐adrenergic agonists and antagonists, demonstrate the presence of functional β‐adrenergic receptors at the SV membrane. Since a recent study shows vH⁺‐ATPase to be upstream of AQP‐6 in the pathway leading from G_αo‐mediated swelling of SV, participation of an endogenous β‐adrenergic agonist, in the binding and stimulation of its receptor to initiate the swelling cascade is demonstrated.     

Average thickness of the gas vesicle wall in Anabaena flos-aquae.

The average thickness of the layer of protein which forms the wall of the gas vesicles in Anabaena flos-aquae was estimated from measurements of their density and geometry. The volume of the gas space in a purified gas vesicle suspension was determined from the contraction which occurred when the gas vesicles were collapsed by pressure. The volume of the protein in the same sample was calculated from its dry weight and density. From knowledge of the geometry of the average gas vesicle the thickness of the protein layer, 1.54 nm, was then calculated. By a similar method the thickness of the Microcystis gas vesicle wall, 1.62 nm, was calculated from data published by others. The average thickness of the protein layer is, as expected, slightly less than the stacking periodicity of collapsed gas vesicle walls indicated by X-ray diffraction studies.Anabaena gas vesicles with a mean length of 494 nm have an average density of 0.119 mg μl^?1 1 mg of protein is present in gas vesicles having a, total volume of 8.43 μl and a gas space of 7.67 μl. Suspensions of isolated gas vesicles with a gas space concentration of 1 μl ml^?1 give a pressure-sensitive optical density, E_1cm (500 nm) of 2.72, but gas vacuoles in cells give a smaller value.     

Effect of an overproduction of accessory Gvp proteins on gas vesicle formation in Haloferax volcanii

Gas vesicle formation in haloarchaea requires the expression of the p-vac region consisting of 14 genes, gvpACNO and gvpDEFGHIJKLM. Expression of gvpFGHIJKLM leads to essential accessory proteins formed in minor amounts. An overexpression of gvpG, gvpH or gvpM in addition to p-vac inhibited gas vesicle formation, whereas large amounts of all other Gvp proteins did not disturb the synthesis. The unbalanced expression and in particular an aggregation of the overproduced Gvp with other accessory Gvp derived from p-vac could be a reason for the inhibition. Western analyses demonstrated that the hydrophobic GvpM (and GvpJ) indeed form multimers. Fluorescent dots of GvpM–GFP were seen in cells in vivo underlining an aggregation of GvpM. In search for proteins neutralizing the inhibitory effect in case of GvpM, p-vac +pGM^ex, +pHM^ex, +pJM^ex, and +pLM^ex transformants were constructed. The inhibitory effect of GvpM on gas vesicle formation was suppressed by GvpH, GvpJ or GvpL, but not by GvpG. Western analyses demonstrated that pHM^ex and pJM^ex transformants contained additional larger protein bands when probed with an antiserum raised against GvpH or GvpJ, implying interactions. The balanced amount of GvpM–GvpH and GvpM–GvpJ appears to be important during gas vesicle genesis.     

Key residues on microtubule responsible for activation of kinesin ATPase

Microtubule (MT) binding accelerates the rate of ATP hydrolysis in kinesin. To understand the underlying mechanism, using charged‐to‐alanine mutational analysis, we identified two independent sites in tubulin, which are critical for kinesin motility, namely, a cluster of negatively charged residues spanning the helix 11–12 (H11–12) loop and H12 of α‐tubulin, and the negatively charged residues in H12 of β‐tubulin. Mutation in the α‐tubulin‐binding site results in a deceleration of ATP hydrolysis (k_cat), whereas mutation in the β‐tubulin‐binding site lowers the affinity for MTs (K_0.5MT). The residue E415 in α‐tubulin seems to be important for coupling MT binding and ATPase activation, because the mutation at this site results in a drastic reduction in the overall rate of ATP hydrolysis, largely due to a deceleration in the reaction of ADP release. Our results suggest that kinesin binding at a region containing α‐E415 could transmit a signal to the kinesin nucleotide pocket, triggering its conformational change and leading to the release of ADP.     

Solid-State NMR Characterization of Gas Vesicle Structure

Gas vesicles are gas-filled buoyancy organelles with walls that consist almost exclusively of gas vesicle protein A (GvpA). Intact, collapsed gas vesicles from the cyanobacterium Anabaena flos-aquae were studied by solid-state NMR spectroscopy, and most of the GvpA sequence was assigned. Chemical shift analysis indicates a coil-α-β-β-α-coil peptide backbone, consistent with secondary-structure-prediction algorithms, and complementary information about mobility and solvent exposure yields a picture of the overall topology of the vesicle subunit that is consistent with its role in stabilizing an air-water interface.     

Solution structure for an Encephalitozoon cuniculi adrenodoxin‐like protein in the oxidized state

Encephalitozoon cuniculi is a unicellular, obligate intracellular eukaryotic parasite in the Microsporidia family and one of the agents responsible for microsporidosis infections in humans. Like most Microsporidia, the genome of E. cuniculi is markedly reduced and the organism contains mitochondria‐like organelles called mitosomes instead of mitochondria. Here we report the solution NMR structure for a protein physically associated with mitosome‐like organelles in E. cuniculi, the 128‐residue, adrenodoxin‐like protein Ec‐Adx (UniProt ID Q8SV19) in the [2Fe‐2S] ferredoxin superfamily. Oxidized Ec‐Adx contains a mixed four‐strand β‐sheet, β2‐β1‐β4‐β3 (↓↑↑↓), loosely encircled by three α‐helices and two 3₁₀‐helices. This fold is similar to the structure observed in other adrenodoxin and adrenodoxin‐like proteins except for the absence of a fifth anti‐parallel β‐strand next to β3 and the position of α3. Cross peaks are missing or cannot be unambiguously assigned for 20 amide resonances in the ¹H‐¹⁵N HSQC spectrum of Ec‐Adx. These missing residues are clustered primarily in two regions, G48‐V61 and L94‐L98, containing the four cysteine residues predicted to ligate the paramagnetic [2Fe‐2S] cluster. Missing amide resonances in ¹H‐¹⁵N HSQC spectra are detrimental to NMR‐based solution structure calculations because ¹H‐¹H NOE restraints are absent (glass half‐empty) and this may account for the absent β‐strand (β5) and the position of α3 in oxidized Ec‐Adx. On the other hand, the missing amide resonances unambiguously identify the presence, and immediate environment, of the paramagnetic [2Fe‐2S] cluster in oxidized Ec‐Adx (glass half‐full).