A role for FtsA in SPOR‐independent localization of the essential Escherichia coli cell division protein FtsN

FtsN is a bitopic membrane protein and the last essential component to localize to the Escherichia coli cell division machinery, or divisome. The periplasmic SPOR domain of FtsN was previously shown to localize to the divisome in a self‐enhancing manner, relying on the essential activity of FtsN and the peptidoglycan synthesis and degradation activities of FtsI and amidases respectively. Because FtsN has a known role in recruiting amidases and is predicted to stimulate the activity of FtsI, it follows that FtsN initially localizes to division sites in a SPOR‐independent manner. Here, we show that the cytoplasmic and transmembrane domains of FtsN (FtsN_Cyto‐TM) facilitated localization of FtsN independently of its SPOR domain but dependent on the early cell division protein FtsA. In addition, SPOR‐independent localization preceded SPOR‐dependent localization, providing a mechanism for the initial localization of FtsN. In support of the role of FtsN_Cyto‐TM in FtsN function, a variant of FtsN lacking the cytoplasmic domain localized to the divisome but failed to complement an ftsN deletion unless it was overproduced. Simultaneous removal of the cytoplasmic and SPOR domains abolished localization and complementation. These data support a model in which FtsA–FtsN interaction recruits FtsN to the divisome, where it can then stimulate the peptidoglycan remodelling activities required for SPOR‐dependent localization.     

Discovery and Characterization of Three New Escherichia coli Septal Ring Proteins That Contain a SPOR Domain: DamX,DedD, and RlpA

SPOR domains are ∼70 amino acids long and occur in >1,500 proteins identified by sequencing of bacterial genomes. The SPOR domains in the FtsN cell division proteins from Escherichia coli and Caulobacter crescentus have been shown to bind peptidoglycan. Besides FtsN, E. coli has three additional SPOR domain proteins—DamX, DedD, and RlpA. We show here that all three of these proteins localize to the septal ring in E. coli. The loss of DamX or DedD either alone or in combination with mutations in genes encoding other division proteins resulted in a variety of division phenotypes, demonstrating that DamX and DedD participate in cytokinesis. In contrast, RlpA mutants divided normally. Follow-up studies revealed that the SPOR domains themselves localize to the septal ring in vivo and bind peptidoglycan in vitro. Even SPOR domains from heterologous organisms, including Aquifex aeolicus, localized to septal rings when produced in E. coli and bound to purified E. coli peptidoglycan sacculi. We speculate that SPOR domains localize to the division site by binding preferentially to septal peptidoglycan. We further suggest that SPOR domain proteins are a common feature of the division apparatus in bacteria. DamX was characterized further and found to interact with multiple division proteins in a bacterial two-hybrid assay. One interaction partner is FtsQ, and several synthetic phenotypes suggest that DamX is a negative regulator of FtsQ function.Cell division in Escherichia coli is mediated by a collection of approximately 20 proteins, all of which localize to the midcell, where they form a structure called the septal ring, or divisome. About half of these proteins are essential for cell division. The corresponding temperature-sensitive mutants or depletion strains become filamentous and die under nonpermissive conditions. The remaining proteins are not essential under most laboratory conditions. In some cases null mutations reveal modest division defects, but in other cases division defects become apparent only under certain growth conditions or in combination with mutations in genes for other division proteins. For reviews of this topic, see references 18, 22, 29, and 67.One of the essential cell division proteins is a bitopic membrane protein named FtsN (see Fig. ​Fig.1A)1A) (13, 14). How FtsN facilitates cell division is not clear. Because overproduction of FtsN rescues a variety of mutants with lesions in genes for other cell division proteins [ftsA(Ts), ftsI(Ts) ftsQ(Ts), ftsEX null, ftsK null, and ftsP (sufI) null strains], it seems likely that one function of FtsN is to improve the assembly and/or stability of the septal ring (13, 20, 24, 30, 58, 63). Very recent evidence indicates that FtsN plays an important role in triggering constriction, probably by allosteric activation of some other component of the septal ring (26).Open in a separate windowFIG. 1.SPOR domain proteins included in this study. (A) Membrane topology and number of amino acids in each domain as retrieved from UniProt release 15.7 (http://www.uniprot.org) or the GTOP update of 15 December 2008 (http://spock.genes.nig.ac.jp/∼genome/gtop.html). N, amino terminus; CM, cytoplasmic membrane; OM, outer membrane. RlpA and VPA1294 have a covalently attached lipid at their amino termini. (B) Multiple-sequence alignment of SPOR domains shown in the present study to localize to the septal ring of E. coli. Sequences were aligned manually to the position-specific scoring matrix (PSSM) from http://www.ncbi.nlm.nih.gov/Class/Structure/pssm/pssm_viewer.cgi with the SPOR domain (Pfam accession no. 05036) as the PSSM identifier (PSSM ID). Residues with identity to those in the consensus sequence from the PSSM alignment are shaded gray. Numbers to the left refer to the first positions of the SPOR domains in the indicated proteins.A notable feature of FtsN is that it contains at its C terminus a peptidoglycan (PG) binding domain known as a SPOR domain (Pfam accession no. 05036) (23, 65, 72). SPOR domains are both common and widespread in bacteria. At the time of this writing (August 2009), over 1,500 proteins that contain a SPOR domain are listed in the Pfam database (23). These proteins come from over 500 bacterial species. The domain is named after the founding member of the protein family, a Bacillus subtilis protein named CwlC that is produced relatively late in the process of sporulation (41). CwlC, which comprises an N-terminal amidase domain and a C-terminal SPOR domain, facilitates release of the mature spore by degrading PG in the mother cell (48, 61).Our interest in SPOR domain proteins was piqued during a study of Vibrio parahaemolyticus (in collaboration with Linda McCarter) when we observed that a gene of unknown function, designated vpa1294, is highly induced in V. parahaemolyticus swarmer cells. The VPA1294 protein was annotated as a “putative DamX-related protein” (44; http://genome.gen-info.osaka-u.ac.jp/bacteria/vpara/). To learn about DamX, we turned to the EcoGene website (http://ecogene.org/) (57), which noted that (i) DamX from E. coli has an essentially unknown function, (ii) overproduction of DamX inhibits cell division (43), and (iii) DamX is one of four E. coli proteins that contain a SPOR domain, the others being the cell division protein FtsN and two proteins of unknown function, DedD and RlpA. Based on this information, we decided to investigate whether DamX, DedD, and RlpA are involved in cell division in E. coli. While this work was in progress, the Thanbichler laboratory demonstrated that Caulobacter crescentus has an FtsN-like protein that is needed for cell division (49) and the de Boer laboratory published a report on DamX, DedD, and RlpA from E. coli (26). We also learned that J. Maddock''s laboratory has been investigating DamX, DedD, and RlpA from E. coli (personal communication). Importantly, the major findings from all four laboratories are in general agreement: SPOR domain proteins are widespread in bacteria, many of these proteins are involved in cell division, and SPOR domains are sufficient for septal localization, probably because SPOR domains bind to septal PG.     

Intragenic Suppressor of a Plug Deletion Nonmotility Mutation in PotB,a Chimeric Stator Protein of Sodium-Driven Flagella

The torque of bacterial flagellar motors is generated by interactions between the rotor and the stator and is coupled to the influx of H⁺ or Na⁺ through the stator. A chimeric protein, PotB, in which the N-terminal region of Vibrio alginolyticus PomB was fused to the C-terminal region of Escherichia coli MotB, can function with PomA as a Na⁺-driven stator in E. coli. Here, we constructed a deletion variant of PotB (with a deletion of residues 41 to 91 [Δ41–91], called PotBΔL), which lacks the periplasmic linker region including the segment that works as a “plug” to inhibit premature ion influx. This variant did not confer motile ability, but we isolated a Na⁺-driven, spontaneous suppressor mutant, which has a point mutation (R109P) in the MotB/PomB-specific α-helix that connects the transmembrane and peptidoglycan binding domains of PotBΔL in the region of MotB. Overproduction of the PomA/PotBΔL(R109P) stator inhibited the growth of E. coli cells, suggesting that this stator has high Na⁺-conducting activity. Mutational analyses of Arg109 and nearby residues suggest that the structural alteration in this α-helix optimizes PotBΔL conformation and restores the proper arrangement of transmembrane helices to form a functional channel pore. We speculate that this α-helix plays a key role in assembly-coupled stator activation.     

Differences in the Structure and Dynamics of the Apo- and Palmitate-ligated Forms of Aedes aegypti Sterol Carrier Protein 2 (AeSCP-2)

Sterol carrier protein-2 (SCP-2) is a nonspecific lipid-binding protein expressed ubiquitously in most organisms. Knockdown of SCP-2 expression in mosquitoes has been shown to result in high mortality in developing adults and significantly lowered fertility. Thus, it is of interest to determine the structure of mosquito SCP-2 and to identify its mechanism of lipid binding. We report here high quality three-dimensional solution structures of SCP-2 from Aedes aegypti determined by NMR spectroscopy in its ligand-free state (AeSCP-2) and in complex with palmitate. Both structures have a similar mixed α/β fold consisting of a five-stranded β-sheet and four α-helices arranged on one side of the β-sheet. Ligand-free AeSCP-2 exhibited regions of structural heterogeneity, as evidenced by multiple two-dimensional ¹⁵N heteronuclear single-quantum coherence peaks for certain amino acids; this heterogeneity disappeared upon complex formation with palmitate. The binding of palmitate to AeSCP-2 was found to decrease the backbone mobility of the protein but not to alter its secondary structure. Complex formation is accompanied by chemical shift differences and a loss of mobility for residues in the loop between helix αI and strand βA. The structural differences between the αI and βA of the mosquito and the vertebrate SCP-2s may explain the differential specificity (insect versus vertebrate) of chemical inhibitors of the mosquito SCP-2.     

Structural and Biochemical Analyses of Mycobacterium tuberculosis N-Acetylmuramyl-l-alanine Amidase Rv3717 Point to a Role in Peptidoglycan Fragment Recycling

Peptidoglycan hydrolases are key enzymes in bacterial cell wall homeostasis. Understanding the substrate specificity and biochemical activity of peptidoglycan hydrolases in Mycobacterium tuberculosis is of special interest as it can aid in the development of new cell wall targeting therapeutics. In this study, we report biochemical and structural characterization of the mycobacterial N-acetylmuramyl-l-alanine amidase, Rv3717. The crystal structure of Rv3717 in complex with a dipeptide product shows that, compared with previously characterized peptidoglycan amidases, the enzyme contains an extra disulfide-bonded β-hairpin adjacent to the active site. The structure of two intermediates in assembly reveal that Zn²⁺ binding rearranges active site residues, and disulfide formation promotes folding of the β-hairpin. Although Zn²⁺ is required for hydrolysis of muramyl dipeptide, disulfide oxidation is not required for activity on this substrate. The orientation of the product in the active site suggests a role for a conserved glutamate (Glu-200) in catalysis; mutation of this residue abolishes activity. The product binds at the head of a closed tunnel, and the enzyme showed no activity on polymerized peptidoglycan. These results point to a potential role for Rv3717 in peptidoglycan fragment recycling.     

Kinesin-5 Allosteric Inhibitors Uncouple the Dynamics of Nucleotide,Microtubule, and Neck-Linker Binding Sites

Kinesin motor domains couple cycles of ATP hydrolysis to cycles of microtubule binding and conformational changes that result in directional force and movement on microtubules. The general principles of this mechanochemical coupling have been established; however, fundamental atomistic details of the underlying allosteric mechanisms remain unknown. This lack of knowledge hampers the development of new inhibitors and limits our understanding of how disease-associated mutations in distal sites can interfere with the fidelity of motor domain function. Here, we combine unbiased molecular-dynamics simulations, bioinformatics analysis, and mutational studies to elucidate the structural dynamic effects of nucleotide turnover and allosteric inhibition of the kinesin-5 motor. Multiple replica simulations of ATP-, ADP-, and inhibitor-bound states together with network analysis of correlated motions were used to create a dynamic protein structure network depicting the internal dynamic coordination of functional regions in each state. This analysis revealed the intervening residues involved in the dynamic coupling of nucleotide, microtubule, neck-linker, and inhibitor binding sites. The regions identified include the nucleotide binding switch regions, loop 5, loop 7, α4-α5-loop 13, α1, and β4-β6-β7. Also evident were nucleotide- and inhibitor-dependent shifts in the dynamic coupling paths linking functional sites. In particular, inhibitor binding to the loop 5 region affected β-sheet residues and α1, leading to a dynamic decoupling of nucleotide, microtubule, and neck-linker binding sites. Additional analyses of point mutations, including P131 (loop 5), Q78/I79 (α1), E166 (loop 7), and K272/I273 (β7) G325/G326 (loop 13), support their predicted role in mediating the dynamic coupling of distal functional surfaces. Collectively, our results and approach, which we make freely available to the community, provide a framework for explaining how binding events and point mutations can alter dynamic couplings that are critical for kinesin motor domain function.     

A Compare-and-Contrast NMR Dynamics Study of Two Related RRMs: U1A and SNF

The U1A/U2B″/SNF family of small nuclear ribonucleoproteins uses a phylogenetically conserved RNA recognition motif (RRM1) to bind RNA stemloops in U1 and/or U2 small nuclear RNA (snRNA). RRMs are characterized by their α/β sandwich topology, and these RRMs use their β-sheet as the RNA binding surface. Unique to this RRM family is the tyrosine-glutamine-phenylalanine (YQF) triad of solvent-exposed residues that are displayed on the β-sheet surface; the aromatic residues form a platform for RNA nucleobases to stack. U1A, U2B″, and SNF have very different patterns of RNA binding affinity and specificity, however, so here we ask how YQF in Drosophila SNF RRM1 contributes to RNA binding, as well as to domain stability and dynamics. Thermodynamic double-mutant cycles using tyrosine and phenylalanine substitutions probe the communication between those two residues in the free and bound states of the RRM. NMR experiments follow corresponding changes in the glutamine side-chain amide in both U1A and SNF, providing a physical picture of the RRM1 β-sheet surface. NMR relaxation and dispersion experiments compare fast (picosecond to nanosecond) and intermediate (microsecond-to-millisecond) dynamics of U1A and SNF RRM1. We conclude that there is a network of amino acid interactions involving Tyr-Gln-Phe in both SNF and U1A RRM1, but whereas mutations of the Tyr-Gln-Phe triad result in small local responses in U1A, they produce extensive microsecond-to-millisecond global motions throughout SNF that alter the conformational states of the RRM.     

Aromatic cluster mutations produce focal modulations of β-sheet structure

Site-directed mutagenesis is a powerful tool for altering the structure and function of proteins in a focused manner. Here, we examined how a model β-sheet protein could be tuned by mutation of numerous surface-exposed residues to aromatic amino acids. We designed these aromatic side chain “clusters” at highly solvent-exposed positions in the flat, single-layer β-sheet of Borrelia outer surface protein A (OspA). This unusual β-sheet scaffold allows us to interrogate the effects of these mutations in the context of well-defined structure but in the absence of the strong scaffolding effects of globular protein architecture. We anticipated that the introduction of a cluster of aromatic amino acid residues on the β-sheet surface would result in large conformational changes and/or stabilization and thereby provide new means of controlling the properties of β-sheets. Surprisingly, X-ray crystal structures revealed that the introduction of aromatic clusters produced only subtle conformational changes in the OspA β-sheet. Additionally, despite burying a large degree of hydrophobic surface area, the aromatic cluster mutants were slightly less stable than the wild-type scaffold. These results thereby demonstrate that the introduction of aromatic cluster mutations can serve as a means for subtly modulating β-sheet conformation in protein design.     

Self-Enhanced Accumulation of FtsN at Division Sites and Roles for Other Proteins with a SPOR Domain (DamX,DedD, and RlpA) in Escherichia coli Cell Constriction

Of the known essential division proteins in Escherichia coli, FtsN is the last to join the septal ring organelle. FtsN is a bitopic membrane protein with a small cytoplasmic portion and a large periplasmic one. The latter is thought to form an α-helical juxtamembrane region, an unstructured linker, and a C-terminal, globular, murein-binding SPOR domain. We found that the essential function of FtsN is accomplished by a surprisingly small essential domain (^EFtsN) of at most 35 residues that is centered about helix H2 in the periplasm. ^EFtsN contributed little, if any, to the accumulation of FtsN at constriction sites. However, the isolated SPOR domain (^SFtsN) localized sharply to these sites, while SPOR-less FtsN derivatives localized poorly. Interestingly, localization of ^SFtsN depended on the ability of cells to constrict and, thus, on the activity of ^EFtsN. This and other results suggest that, compatible with a triggering function, FtsN joins the division apparatus in a self-enhancing fashion at the time of constriction initiation and that its SPOR domain specifically recognizes some form of septal murein that is only transiently available during the constriction process. SPOR domains are widely distributed in bacteria. The isolated SPOR domains of three additional E. coli proteins of unknown function, DamX, DedD, and RlpA, as well as that of Bacillus subtilis CwlC, also accumulated sharply at constriction sites in E. coli, suggesting that septal targeting is a common property of SPORs. Further analyses showed that DamX and, especially, DedD are genuine division proteins that contribute significantly to the cell constriction process.Bacterial cytokinesis is mediated by a ring-shaped apparatus. Assembly of this septal ring (SR; also called the divisome or septasome) begins at the future site of fission, well before cell constriction initiates, and it remains associated with the leading edge of the invaginating cell envelope until fission is completed. The mature ring in Escherichia coli is made up of at least 10 essential division proteins (FtsA, -B, -I, -K, -L, -N, -Q, -W, and -Z and ZipA), which are each needed to prevent a lethal filamentation phenotype. The first known step in assembly of the division apparatus is polymerization of FtsZ just underneath the cytoplasmic membrane. These polymers are joined by FtsA and ZipA via direct interactions with FtsZ, resulting in an intermediate ring structure (the Z ring), onto which the remaining components assemble in a specific order to form a constriction-competent complex.In addition to the essential SR proteins, a growing number of nonessential proteins that associate with the organelle are being identified. Some of the latter are likely to serve redundant functions, while some may be required only under particular conditions (for reviews on the topic, see references 15, 19, and 25).FtsN belongs to the essential SR proteins and is thought to be the last of this class to join the organelle before the onset of cell constriction (1, 9, 11, 57, 59). It is a type II bitopic transmembrane species of 319 residues with a small cytoplasmic domain (residues 1 to 30), a single transmembrane domain (residues 31 to 54), and a large periplasmic domain (residues 55 to 319) (12) (Fig. ​(Fig.1).1). The periplasmic domain comprises three short regions with an α-helical character that are centered around residues 62 to 67 (H1), 80 to 93 (H2), and 117 to 123 (H3), an unstructured glutamine-rich linker (residues 124 to 242), and a C-terminal globular SPOR domain (residues 243 to 319) that has an affinity for peptidoglycan (55, 60) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.E. coli ftsN locus, FtsN domains, and properties of genetic constructs. Shown are the EZTnKan-2 insertion site in ftsN^slm117 strains and the deletion-replacement in ftsN<>aph (ΔftsN) strains. Numbers refer to the site of insertion (black triangle) or to the base pairs that were replaced with an aph cassette (doubleheaded arrow), counting from the start of ftsN. The domain structure of FtsN is illustrated below the ftsN gene. Indicated are the transmembrane domain (TM; light gray), helices H1, H2, and H3 (black) in the periplasmic juxtamembrane region, and the C-terminal SPOR domain (^SFtsN; dark gray). The small periplasmic peptide that is sufficient for FtsN′s essential function in cell division (^EFtsN [see text]) is indicated with the doubleheaded arrow below the domain structure diagram. Also shown are inserts present on plasmids that produce fusions of various portions of FtsN to GFP or ^TTGFP under the control of the P_lac regulatory region. ^TTGFP-fusions contain the TorA signal peptide (hatched box) that is cleaved upon export to the periplasm via the twin arginine transport (Tat) system. Columns indicate the FtsN residues present in each fusion, whether the fusion could (+) or could not (−) compensate for the absence of native FtsN, and whether it accumulated at constriction sites sharply (+++) or poorly (−−+) or appeared evenly distibuted along the periphery of the cell (−−−).As with most SR proteins, it is unclear what the essential role of FtsN is. The ftsN gene was first identified as a multicopy suppressor of a Ts allele in essential division gene ftsA (11). Elevated levels of FtsN were subsequently found to also suppress some Ts alleles in ftsI, ftsK, and ftsQ (11, 18), and even to allow the propagation of cells with a complete lack of FtsK (22, 26) or of FtsEX (48). Depletion of FtsN allows assembly of all the other known essential components into nonconstricting SRs, but the number of ring structures per unit of cell length in FtsN⁻ filaments is two- to threefold lower than in wild-type (WT) cells (9). Bacterial two-hybrid studies suggest that FtsN interacts with several other SR proteins, including FtsA, FtsI (penicillin-binding protein 3 [PBP3]), FtsQ, FtsW, and MtgA (10, 16, 17, 38). Moreover, it was recently shown that the requirement for FtsN itself can be bypassed in cells producing certain mutant forms of FtsA, which are thought to stabilize the SR to a greater degree than native FtsA (5). These observations are all compatible with a general role of FtsN in stabilizing the ring structure. In addition, it was recently found that FtsN interacts directly with PBP1B, one of the major bifunctional murein synthases in E. coli, and that it can stimulate both its transglycosylase and transpeptidase activities in vitro (46). Thus, in addition to stabilizing the SR, FtsN may have a more specific role in modulating septal murein synthesis. Lastly, based on the fact that FtsN is the last known essential protein to join the SR, it is attractive to speculate the protein plays a role in triggering the constriction phase (10, 25). To what degree any of these proposed functions contribute to the essentiality of FtsN remains unclear.What does seem clear is that the essential activity of FtsN takes place in the periplasm and that residues 139 to 319 are dispensable for its essential function (12, 55). In addition, as residues 1 to 45 are also dispensable for targeting of FtsN to division sites, some portion of the periplasmic domain must also be sufficient to direct the protein to the division apparatus (1).In a genetic screen for synthetic lethality with min (slm) (6, 7), we isolated a mutant strain carrying a transposon insertion in codon 119 of ftsN. The viability of cells containing this severely truncated ftsN^slm117 allele prompted us to better define the functional domains of FtsN, and we did so by studying the properties of fusions between various portions of FtsN to green fluorescent protein (GFP). To sublocalize a subset of these, we took advantage of the ability of the twin arginine transport system (Tat) to export functional and fluorescent GFP fusions into the periplasm, such that their periplasmic localization could be determined in live cells by fluorescence microscopy (6, 8, 50, 54).We show that the essential function of FtsN can be performed by a surprisingly small periplasmic peptide of at most 35 residues that is centered around helix H2 but that this essential domain (^EFtsN) itself is unlikely to contribute much, if anything, to the accumulation of FtsN at constriction sites. On the other hand, the nonessential periplasmic SPOR domain (^SFtsN) localized sharply to these sites by itself, while SPOR-less FtsN derivatives localized poorly, at best. Notably, septal localization of ^SFtsN depended on coproduction of ^EFtsN, in cis or in trans, unless cells were provided with the FtsA^E124A protein (5) to allow constriction to ensue in the complete absence of ^EFtsN. Localization of ^SFtsN also depended on the activity of FtsI (PBP3) and the presence of at least one of the periplasmic murein amidases, AmiA, -B, or -C. The results suggest that FtsN joins the division apparatus in a self-enhancing fashion at the time of constriction initiation, which is compatible with a role of the protein in triggering the constriction phase of the division process. In addition, the results, taken together with earlier biochemical work (44, 46, 55), suggest that ^SFtsN is recruited to some form of septal murein that accumulates only transiently at sites of active constriction.In addition to FtsN, E. coli produces three proteins of unknown function that also bear a C-terminal SPOR domain (PF05036; Pfam 23) (20). Two of these, DamX and DedD, are inner membrane proteins with the same topology as FtsN, while the third, RlpA, is an outer membrane lipoprotein (43, 47, 53). We found that all three also accumulate at septal rings and that each of their SPOR domains act as autonomous septal targeting determinants. Moreover, phenotypes of the mutants indicate that both DamX and DedD contribute to the cell constriction process, leading to classification of these proteins as new nonessential division proteins.A SPOR domain is predicted to be present in at least 1,650 (putative) proteins from over 500 bacterial species (PF05036; Pfam 23) (20), raising the question as to how far SPOR properties have been conserved. We find that the SPOR domain of CwlC, a Bacillus subtilis murein amidase that is active during late stages of sporulation (39, 44), also accumulates sharply at division sites in E. coli.Our results predict that many other bacterial SPOR domain proteins specifically recognize the same or closely related target molecule(s) that accumulates transiently at sites of cell constriction. This is supported by a very recent study showing that SPOR domain proteins from Burkholderia thailandensis, Caulobacter crescentus, and Myxococcus xanthus accumulate at cell constriction sites as well (45).     

Site-directed mutagenesis analysis of the structural interaction of the single-strand-break repair protein,X-ray cross-complementing group 1, with DNA polymerase beta

Human X-ray cross-complementing group 1 (XRCC1) is a single-strand DNA break repair protein which forms a base excision repair (BER) complex with DNA polymerase β (β-Pol). Here we report a site- directed mutational analysis in which 16 mutated versions of the XRCC1 N-terminal domain (XRCC1-NTD) were constructed on the basis of previous NMR results that had implicated the proximity of various surface residues to β-Pol. Mutant proteins defective in XRCC1-NTD interaction with β-Pol and with a β-Pol–gapped DNA complex were determined by gel filtration chromatography and a gel mobility shift assay. The interaction surface determined from the mutated residues was found to encompass β-strand D and E of the five-stranded β-sheet (βABGDE) and the protruding α2 helix of the XRCC1-NTD. Mutations that included F67A (βD), E69K (βD), V86R (βE) on the five-stranded β-sheet and deletion of the α2 helix, but not mutations within α2, abolished binding of the XRCC1-NTD to β-Pol. A Y136A mutant abolished β-Pol binding, and a R109S mutant reduced β-Pol binding. E98K, E98A, N104A, Y136A, R109S, K129E, F142A, R31A/K32A/R34A and δ-helix-2 mutants displayed temperature dependent solubility. These findings confirm the importance of the α2 helix and the βD and βE strands of XRCC1-NTD to the energetics of β-Pol binding. Establishing the direct contacts in the β-Pol XRCC1 complex is a critical step in understanding how XRCC1 fulfills its numerous functions in DNA BER.     

Computational analyses of protein coded by rice (Oryza sativa japonica) cDNA (GI: 32984786) indicate lectin like Ca2+ binding properties for Eicosapenta Peptide Repeats (EPRs)

Eicosapenta peptide repeats (EPRs) occur exclusively in flowering plant genomes and exhibit very high amino acid residue conservation across occurrence. DNA and amino acid sequence searches yielded no indications about the function due to absence of similarity to known sequences. Tertiary structure of an EPR protein coded by rice (Oryza sativa japonica) cDNA (GI: 32984786) was determined based on ab initio methodology in order to draw clues on functional significance of EPRs. The resultant structure comprised of seven α-helices and thirteen anti-parallel β-sheets. Surface-mapping of conserved residues onto the structure deduced that (i) regions equivalent to β α4- the primary function of EPR protein could be Ca²⁺ binding, and (iii) the putative EPR Ca²⁺ binding domain is structurally similar to calcium-binding domains of plant lectins. Additionally, the phylogenetic analysis showed an evolving taxa-specific distribution of EPR proteins observed in some GNA-like lectins.     

Evolution rescues folding of human immunodeficiency virus-1 envelope glycoprotein GP120 lacking a conserved disulfide bond

The majority of eukaryotic secretory and membrane proteins contain disulfide bonds, which are strongly conserved within protein families because of their crucial role in folding or function. The exact role of these disulfide bonds during folding is unclear. Using virus-driven evolution we generated a viral glycoprotein variant, which is functional despite the lack of an absolutely conserved disulfide bond that links two antiparallel β-strands in a six-stranded β-barrel. Molecular dynamics simulations revealed that improved hydrogen bonding and side chain packing led to stabilization of the β-barrel fold, implying that β-sheet preference codirects glycoprotein folding in vivo. Our results show that the interactions between two β-strands that are important for the formation and/or integrity of the β-barrel can be supported by either a disulfide bond or β-sheet favoring residues.     

Identification of a Polar Region in Transmembrane Domain 6 That Regulates the Function of the G Protein-Coupled α-Factor Receptor

The α-factor pheromone receptor (Ste2p) of the yeast Saccharomyces cerevisiae belongs to the family of G protein-coupled receptors that contain seven transmembrane domains (TMDs). Because polar residues can influence receptor structure by forming intramolecular contacts between TMDs, we tested the role of the five polar amino acids in TMD6 of the α-factor receptor by mutating these residues to nonpolar leucine. Interestingly, a subset of these mutants showed increased affinity for ligand and constitutive receptor activity. The mutation of the most polar residue, Q253L, resulted in 25-fold increased affinity and a 5-fold-higher basal level of signaling that was equal to about 19% of the α-factor induced maximum signal. Mutation of the adjacent residue, S254L, caused weaker constitutive activity and a 5-fold increase in affinity. Comparison of nine different mutations affecting Ser²⁵⁴ showed that an S254F mutation caused higher constitutive activity, suggesting that a large hydrophobic amino acid residue at position 254 alters transmembrane helix packing. Thus, these studies indicate that Gln²⁵³ and Ser²⁵⁴ are likely to be involved in intramolecular interactions with other TMDs. Furthermore, Gln²⁵³ and Ser²⁵⁴ fall on one side of the transmembrane helix that is on the opposite side from residues that do not cause constitutive activity when mutated. These results suggest that Gln²⁵³ and Ser²⁵⁴ face inward toward the other TMDs and thus provide the first experimental evidence to suggest the orientation of a TMD in this receptor. Consistent with this, we identified two residues in TMD7 (Ser²⁸⁸ and Ser²⁹²) that are potential contact residues for Gln²⁵³ because mutations affecting these residues also cause constitutive activity. Altogether, these results identify a new domain of the α-factor receptor that regulates its ability to enter the activated conformation.     

Expression, purification, and structural characterization of CfrA, a putative iron transporter from Campylobacter jejuni

The gene for the Campylobacter ferric receptor (CfrA), a putative iron-siderophore transporter in the enteric food-borne pathogen Campylobacter jejuni, was cloned, and the membrane protein was expressed in Escherichia coli, affinity purified, and then reconstituted into model lipid membranes. Fourier transform infrared spectra recorded from the membrane-reconstituted CfrA are similar to spectra that have been recorded from other iron-siderophore transporters and are highly characteristic of a β-sheet protein (~44% β-sheet and ~10% α-helix). CfrA undergoes relatively extensive peptide hydrogen-deuterium exchange upon exposure to ²H₂O and yet is resistant to thermal denaturation at temperatures up to 95°C. The secondary structure, relatively high aqueous solvent exposure, and high thermal stability are all consistent with a transmembrane β-barrel structure containing a plug domain. Sequence alignments indicate that CfrA contains many of the structural motifs conserved in other iron-siderophore transporters, including the Ton box, PGV, IRG, RP, and LIDG motifs of the plug domain. Surprisingly, a homology model reveals that regions of CfrA that are expected to play a role in enterobactin binding exhibit sequences that differ substantially from the sequences of the corresponding regions that play an essential role in binding/transport by the E. coli enterobactin transporter, FepA. The sequence variations suggest that there are differences in the mechanisms used by CfrA and FepA to interact with bacterial siderophores. It may be possible to exploit these structural differences to develop CfrA-specific therapeutics.     

Model of β-Sheet of Muscle Fatty Acid Binding Protein of Locusta migratoria Displays Characteristic Topology

The β-sheet of muscle fatty acid binding protein of Locusta migratoria (Lm-FABP) was modeled by employing 2-D NMR data and the Rigid Body Assembly method. The model shows the β-sheet to comprise ten β-strands arranged anti-parallel to each other. There is a β-bulge between Ser 13 and Gln 14 which is a difference from the published structure of β-sheet of bovine heart Fatty Acid Binding Protein. Also, a hydrophobic patch consisting of Ile 45, Phe 51, Phe 64 and Phe 66 is present on the surface which is characteristic of most Fatty Acid Binding Proteins. A “gap” is present between βD and βE that provides evidence for the presence of a portal or opening between the polypeptide chains which allows ligand fatty acids to enter the protein cavity and bind to the protein.     

How Bacteria Consume Their Own Exoskeletons (Turnover and Recycling of Cell Wall Peptidoglycan)

Summary: The phenomenon of peptidoglycan recycling is reviewed. Gram-negative bacteria such as Escherichia coli break down and reuse over 60% of the peptidoglycan of their side wall each generation. Recycling of newly made peptidoglycan during septum synthesis occurs at an even faster rate. Nine enzymes, one permease, and one periplasmic binding protein in E. coli that appear to have as their sole function the recovery of degradation products from peptidoglycan, thereby making them available for the cell to resynthesize more peptidoglycan or to use as an energy source, have been identified. It is shown that all of the amino acids and amino sugars of peptidoglycan are recycled. The discovery and properties of the individual proteins and the pathways involved are presented. In addition, the possible role of various peptidoglycan degradation products in the induction of β-lactamase is discussed.     

Structural Transitions and Interactions in the Early Stages of Human Glucagon Amyloid Fibrillation

A mechanistic understanding of the intermolecular interactions and structural changes during fibrillation is crucial for the design of safe and efficacious glucagon formulations. Amide hydrogen/deuterium exchange with mass spectrometric analysis was used to identify the interactions and amino acids involved in the initial stages of glucagon fibril formation at acidic pH. Kinetic measurements from intrinsic and thioflavin T fluorescence showed sigmoidal behavior. Secondary structural measurement of fibrillating glucagon using far-UV circular dichroism spectroscopy showed changes in structure from random coil → α-helix → β-sheet, with increase in α-helix content during the lag phase followed by increase in β-sheet content during the growth phase. Hydrogen/deuterium exchange with mass spectrometric analysis of fibrillating glucagon suggested that C-terminal residues 22–29 are involved in interactions during the lag phase, during which N-terminal residues 1–6 showed no changes. Molecular dynamics simulations of glucagon fragments showed C-terminal to C-terminal interactions with greater α-helix content for the 20–29 fragment, with hydrophobic and aromatic residues (Phe-22, Trp-25, Val-23, and Met-27) predominantly involved. Overall, the study shows that glucagon interactions during the early phase of fibrillation are mediated through C-terminal residues, which facilitate the formation of α-helix-rich oligomers, which further undergo structural rearrangement and elongation to form β-sheet-rich mature fibrils.     

Three-dimensional structure of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum at 2.9 A resolution

The three-dimensional structure of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Rhodospirillum rubrum has been determined at 2.9 Å resolution by X-ray crystallographic methods. The MIR-electron density map was substantially improved by two-fold non-crystallographic symmetry averaging. The polypeptide chains in the dimer were traced using a graphics display system with the help of the BONES option in FRODO. The dimer has approximate dimensions of 50 x 72 x 105 Å. The enzyme subunit is a typical two-domain protein. The smaller, N-terminal domain consists of 137 amino acid residues and forms a central, mixed five-stranded β-sheet with α-helices on both sides of the sheet. The larger C-terminal domain consists of 329 amino acid residues. This domain has an eight-stranded parallel α/β barrel structure as found in triosephosphate isomerase and a number of other functionally non-related proteins. The active site in Rubisco determined by difference Fourier techniques and fitting of active site residues to the electron density map, is located at the carboxy-end of the β-strands in the α/β barrel of the C-terminal domain. There are few domain–domain interactions within the subunit. The interactions at the interface between the two subunits of the dimer are tight and extensive. There are tight contacts between the two C-terminal domains, which build up the core of the molecule. There are also interactions between the N-terminal domain of one subunit and the C-terminal domain of the second subunit, close to the active site.