Publications by authors named "Maria A Schumacher"

90 Publications

Evolution of a σ-(c-di-GMP)-anti-σ switch.

Proc Natl Acad Sci U S A 2021 Jul;118(30)

Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, United Kingdom;

Filamentous actinobacteria of the genus have a complex lifecycle involving the differentiation of reproductive aerial hyphae into spores. We recently showed c-di-GMP controls this transition by arming a unique anti-σ, RsiG, to bind the sporulation-specific σ, WhiG. The RsiG-(c-di-GMP)-WhiG structure revealed that a monomeric RsiG binds c-di-GMP via two E(X)S(X)R(X)Q(X)D repeat motifs, one on each helix of an antiparallel coiled-coil. Here we show that RsiG homologs are found scattered throughout the Actinobacteria. Strikingly, RsiGs from unicellular bacteria descending from the most basal branch of the Actinobacteria are small proteins containing only one c-di-GMP binding motif, yet still bind their WhiG partners. Our structure of a (RsiG)-(c-di-GMP)-WhiG complex revealed that these single-motif RsiGs are able to form an antiparallel coiled-coil through homodimerization, thereby allowing them to bind c-di-GMP similar to the monomeric twin-motif RsiGs. Further data show that in the unicellular actinobacterium , the (RsiG)-(c-di-GMP)-WhiG regulatory switch controls type IV pilus expression. Phylogenetic analysis indicates the single-motif RsiGs likely represent the ancestral state and an internal gene-duplication event gave rise to the twin-motif RsiGs inherited elsewhere in the Actinobacteria. Thus, these studies show how the anti-σ RsiG has evolved through an intragenic duplication event from a small protein carrying a single c-di-GMP binding motif, which functions as a homodimer, to a larger protein carrying two c-di-GMP binding motifs, which functions as a monomer. Consistent with this, our structures reveal potential selective advantages of the monomeric twin-motif anti-σ factors.
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http://dx.doi.org/10.1073/pnas.2105447118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8325347PMC
July 2021

Structures of Neisseria gonorrhoeae MtrR-operator complexes reveal molecular mechanisms of DNA recognition and antibiotic resistance-conferring clinical mutations.

Nucleic Acids Res 2021 04;49(7):4155-4170

Department of Biochemistry, Duke University School of Medicine, Durham, NC 27710, USA.

Mutations within the mtrR gene are commonly found amongst multidrug resistant clinical isolates of Neisseria gonorrhoeae, which has been labelled a superbug by the Centers for Disease Control and Prevention. These mutations appear to contribute to antibiotic resistance by interfering with the ability of MtrR to bind to and repress expression of its target genes, which include the mtrCDE multidrug efflux transporter genes and the rpoH oxidative stress response sigma factor gene. However, the DNA-recognition mechanism of MtrR and the consensus sequence within these operators to which MtrR binds has remained unknown. In this work, we report the crystal structures of MtrR bound to the mtrCDE and rpoH operators, which reveal a conserved, but degenerate, DNA consensus binding site 5'-MCRTRCRN4YGYAYGK-3'. We complement our structural data with a comprehensive mutational analysis of key MtrR-DNA contacts to reveal their importance for MtrR-DNA binding both in vitro and in vivo. Furthermore, we model and generate common clinical mutations of MtrR to provide plausible biochemical explanations for the contribution of these mutations to multidrug resistance in N. gonorrhoeae. Collectively, our findings unveil key biological mechanisms underlying the global stress responses of N. gonorrhoeae.
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http://dx.doi.org/10.1093/nar/gkab213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8053128PMC
April 2021

Structural Basis for Virulence Activation of Francisella tularensis.

Mol Cell 2021 01 19;81(1):139-152.e10. Epub 2020 Nov 19.

Department of Biochemistry, Duke University School of Medicine, Durham, NC 27710, USA. Electronic address:

The bacterium Francisella tularensis (Ft) is one of the most infectious agents known. Ft virulence is controlled by a unique combination of transcription regulators: the MglA-SspA heterodimer, PigR, and the stress signal, ppGpp. MglA-SspA assembles with the σ-associated RNAP holoenzyme (RNAPσ), forming a virulence-specialized polymerase. These factors activate Francisella pathogenicity island (FPI) gene expression, which is required for virulence, but the mechanism is unknown. Here we report FtRNAPσ-promoter-DNA, FtRNAPσ-(MglA-SspA)-promoter DNA, and FtRNAPσ-(MglA-SspA)-ppGpp-PigR-promoter DNA cryo-EM structures. Structural and genetic analyses show MglA-SspA facilitates σ binding to DNA to regulate virulence and virulence-enhancing genes. Our Escherichia coli RNAPσhomodimeric EcSspA structure suggests this is a general SspA-transcription regulation mechanism. Strikingly, our FtRNAPσ-(MglA-SspA)-ppGpp-PigR-DNA structure reveals ppGpp binding to MglA-SspA tethers PigR to promoters. PigR in turn recruits FtRNAP αCTDs to DNA UP elements. Thus, these studies unveil a unique mechanism for Ft pathogenesis involving a virulence-specialized RNAP that employs two (MglA-SspA)-based strategies to activate virulence genes.
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http://dx.doi.org/10.1016/j.molcel.2020.10.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7959165PMC
January 2021

DNA mismatches reveal conformational penalties in protein-DNA recognition.

Nature 2020 11 21;587(7833):291-296. Epub 2020 Oct 21.

Center for Genomic and Computational Biology, Duke University School of Medicine, Durham, NC, USA.

Transcription factors recognize specific genomic sequences to regulate complex gene-expression programs. Although it is well-established that transcription factors bind to specific DNA sequences using a combination of base readout and shape recognition, some fundamental aspects of protein-DNA binding remain poorly understood. Many DNA-binding proteins induce changes in the structure of the DNA outside the intrinsic B-DNA envelope. However, how the energetic cost that is associated with distorting the DNA contributes to recognition has proven difficult to study, because the distorted DNA exists in low abundance in the unbound ensemble. Here we use a high-throughput assay that we term SaMBA (saturation mismatch-binding assay) to investigate the role of DNA conformational penalties in transcription factor-DNA recognition. In SaMBA, mismatched base pairs are introduced to pre-induce structural distortions in the DNA that are much larger than those induced by changes in the Watson-Crick sequence. Notably, approximately 10% of mismatches increased transcription factor binding, and for each of the 22 transcription factors that were examined, at least one mismatch was found that increased the binding affinity. Mismatches also converted non-specific sites into high-affinity sites, and high-affinity sites into 'super sites' that exhibit stronger affinity than any known canonical binding site. Determination of high-resolution X-ray structures, combined with nuclear magnetic resonance measurements and structural analyses, showed that many of the DNA mismatches that increase binding induce distortions that are similar to those induced by protein binding-thus prepaying some of the energetic cost incurred from deforming the DNA. Our work indicates that conformational penalties are a major determinant of protein-DNA recognition, and reveals mechanisms by which mismatches can recruit transcription factors and thus modulate replication and repair activities in the cell.
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http://dx.doi.org/10.1038/s41586-020-2843-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7666076PMC
November 2020

When is a transcription factor a NAP?

Curr Opin Microbiol 2020 06 28;55:26-33. Epub 2020 Feb 28.

Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK. Electronic address:

Proteins that regulate transcription often also play an architectural role in the genome. Thus, it has been difficult to define with precision the distinctions between transcription factors and nucleoid-associated proteins (NAPs). Anachronistic descriptions of NAPs as 'histone-like' implied an organizational function in a bacterial chromatin-like complex. Definitions based on protein abundance, regulatory mechanisms, target gene number, or the features of their DNA-binding sites are insufficient as marks of distinction, and trying to distinguish transcription factors and NAPs based on their ranking within regulatory hierarchies or positions in gene-control networks is also unsatisfactory. The terms 'transcription factor' and 'NAP' are ad hoc operational definitions with each protein lying along a spectrum of structural and functional features extending from highly specific actors with few gene targets to those with a pervasive influence on the transcriptome. The Streptomyces BldC protein is used to illustrate these issues.
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http://dx.doi.org/10.1016/j.mib.2020.01.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8048100PMC
June 2020

High-resolution crystal structures of Escherichia coli FtsZ bound to GDP and GTP.

Acta Crystallogr F Struct Biol Commun 2020 Feb 5;76(Pt 2):94-102. Epub 2020 Feb 5.

Department of Biochemistry, Duke University School of Medicine, Box 3711, DUMC, Durham, NC 27710, USA.

Bacterial cytokinesis is mediated by the Z-ring, which is formed by the prokaryotic tubulin homolog FtsZ. Recent data indicate that the Z-ring is composed of small patches of FtsZ protofilaments that travel around the bacterial cell by treadmilling. Treadmilling involves a switch from a relaxed (R) state, favored for monomers, to a tense (T) conformation, which is favored upon association into filaments. The R conformation has been observed in numerous monomeric FtsZ crystal structures and the T conformation in Staphylococcus aureus FtsZ crystallized as assembled filaments. However, while Escherichia coli has served as a main model system for the study of the Z-ring and the associated divisome, a structure has not yet been reported for E. coli FtsZ. To address this gap, structures were determined of the E. coli FtsZ mutant FtsZ(L178E) with GDP and GTP bound to 1.35 and 1.40 Å resolution, respectively. The E. coli FtsZ(L178E) structures both crystallized as straight filaments with subunits in the R conformation. These high-resolution structures can be employed to facilitate experimental cell-division studies and their interpretation in E. coli.
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http://dx.doi.org/10.1107/S2053230X20001132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7010359PMC
February 2020

c-di-GMP Arms an Anti-σ to Control Progression of Multicellular Differentiation in Streptomyces.

Mol Cell 2020 02 3;77(3):586-599.e6. Epub 2019 Dec 3.

Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK. Electronic address:

Streptomyces are our primary source of antibiotics, produced concomitantly with the transition from vegetative growth to sporulation in a complex developmental life cycle. We previously showed that the signaling molecule c-di-GMP binds BldD, a master repressor, to control initiation of development. Here we demonstrate that c-di-GMP also intervenes later in development to control differentiation of the reproductive hyphae into spores by arming a novel anti-σ (RsiG) to bind and sequester a sporulation-specific σ factor (σ). We present the structure of the RsiG-(c-di-GMP)-σ complex, revealing an unusual, partially intercalated c-di-GMP dimer bound at the RsiG-σ interface. RsiG binds c-di-GMP in the absence of σ, employing a novel E(X)S(X)R(X)Q(X)D motif repeated on each helix of a coiled coil. Further studies demonstrate that c-di-GMP is essential for RsiG to inhibit σ. These findings reveal a newly described control mechanism for σ-anti-σ complex formation and establish c-di-GMP as the central integrator of Streptomyces development.
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http://dx.doi.org/10.1016/j.molcel.2019.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005675PMC
February 2020

Structures of MERS1, the 5' processing enzyme of mitochondrial mRNAs in .

RNA 2020 01 8;26(1):69-82. Epub 2019 Nov 8.

Department of Biochemistry, Duke University School of Medicine, Durham, North Carolina 27710, USA.

Most mitochondrial mRNAs are transcribed as polycistronic precursors that are cleaved by endonucleases to produce mature mRNA transcripts. However, recent studies have shown that mitochondrial transcripts in the kinetoplastid protozoan, , are transcribed individually. Also unlike most mitochondrial mRNAs, the 5' end of these transcripts harbor a triphosphate that is hydrolyzed. This modification is carried out by a putative Nudix hydrolase called MERS1. The Nudix motif in MERS1 is degenerate, lacking a conserved glutamic acid, thus it is unclear how it may bind its substrates and whether it contains a Nudix fold. To obtain insight into this unusual hydrolase, we determined structures of apo, GTP-bound and RNA-bound MERS1 to 2.30 Å, 2.45 Å, and 2.60 Å, respectively. The MERS1 structure has a unique fold that indeed contains a Nudix motif. The nucleotide bound structures combined with binding studies reveal that MERS1 shows preference for RNA sequences with a central guanine repeat which it binds in a single-stranded conformation. The apo MERS1 structure indicates that a significant portion of its nucleotide binding site folds upon substrate binding. Finally, a potential interaction region for a binding partner, MERS2, that activates MERS1 was identified. The MERS2-like peptide inserts a glutamate near the missing Nudix acidic residue in the RNA binding pocket, suggesting how the enzyme may be activated. Thus, the combined studies reveal insight into the structure and enzyme properties of MERS1 and its substrate-binding activities.
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http://dx.doi.org/10.1261/rna.072231.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6913127PMC
January 2020

Harnessing calcineurin-FK506-FKBP12 crystal structures from invasive fungal pathogens to develop antifungal agents.

Nat Commun 2019 09 19;10(1):4275. Epub 2019 Sep 19.

Division of Pediatric Infectious Diseases, Department of Pediatrics, Duke University Medical Center, Durham, NC, 27710, USA.

Calcineurin is important for fungal virulence and a potential antifungal target, but compounds targeting calcineurin, such as FK506, are immunosuppressive. Here we report the crystal structures of calcineurin catalytic (CnA) and regulatory (CnB) subunits complexed with FK506 and the FK506-binding protein (FKBP12) from human fungal pathogens (Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans and Coccidioides immitis). Fungal calcineurin complexes are similar to the mammalian complex, but comparison of fungal and human FKBP12 (hFKBP12) reveals conformational differences in the 40s and 80s loops. NMR analysis, molecular dynamic simulations, and mutations of the A. fumigatus CnA/CnB-FK506-FKBP12-complex identify a Phe88 residue, not conserved in hFKBP12, as critical for binding and inhibition of fungal calcineurin. These differences enable us to develop a less immunosuppressive FK506 analog, APX879, with an acetohydrazine substitution of the C22-carbonyl of FK506. APX879 exhibits reduced immunosuppressive activity and retains broad-spectrum antifungal activity and efficacy in a murine model of invasive fungal infection.
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http://dx.doi.org/10.1038/s41467-019-12199-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6753081PMC
September 2019

Infrared Spectroscopic Observation of a G-C Hoogsteen Base Pair in the DNA:TATA-Box Binding Protein Complex Under Solution Conditions.

Angew Chem Int Ed Engl 2019 08 25;58(35):12010-12013. Epub 2019 Jul 25.

Department of Biochemistry, Duke University Medical Center, Durham, NC, 27710, USA.

Hoogsteen DNA base pairs (bps) are an alternative base pairing to canonical Watson-Crick bps and are thought to play important biochemical roles. Hoogsteen bps have been reported in a handful of X-ray structures of protein-DNA complexes. However, there are several examples of Hoogsteen bps in crystal structures that form Watson-Crick bps when examined under solution conditions. Furthermore, Hoogsteen bps can sometimes be difficult to resolve in DNA:protein complexes by X-ray crystallography due to ambiguous electron density and by solution-state NMR spectroscopy due to size limitations. Here, using infrared spectroscopy, we report the first direct solution-state observation of a Hoogsteen (G-C ) bp in a DNA:protein complex under solution conditions with specific application to DNA-bound TATA-box binding protein. These results support a previous assignment of a G-C Hoogsteen bp in the complex, and indicate that Hoogsteen bps do indeed exist under solution conditions in DNA:protein complexes.
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http://dx.doi.org/10.1002/anie.201902693DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719543PMC
August 2019

Structures of maintenance of carboxysome distribution Walker-box McdA and McdB adaptor homologs.

Nucleic Acids Res 2019 06;47(11):5950-5962

Department of Biochemistry, Duke University School of Medicine, Durham, NC 27710, USA.

Carboxysomes, protein-coated organelles in cyanobacteria, are important in global carbon fixation. However, these organelles are present at low copy in each cell and hence must be segregated to ensure transmission from one generation to the next. Recent studies revealed that a DNA partition-like ParA-ParB system mediates carboxysome maintenance, called McdA-McdB. Here, we describe the first McdA and McdB homolog structures. McdA is similar to partition ParA Walker-box proteins, but lacks the P-loop signature lysine involved in ATP binding. Strikingly, a McdA-ATP structure shows that a lysine distant from the P-loop and conserved in McdA homologs, enables ATP-dependent nucleotide sandwich dimer formation. Similar to partition ParA proteins this ATP-bound form binds nonspecific-DNA. McdB, which we show directly binds McdA, harbors a unique fold and appears to form higher-order oligomers like partition ParB proteins. Thus, our data reveal a new signature motif that enables McdA dimer formation and indicates that, similar to DNA segregation, carboxysome maintenance systems employ Walker-box proteins as DNA-binding motors while McdB proteins form higher order oligomers, which could function as adaptors to link carboxysomes and provide for stable transport by the McdA proteins.
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http://dx.doi.org/10.1093/nar/gkz314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6582323PMC
June 2019

The RRM of the kRNA-editing protein TbRGG2 uses multiple surfaces to bind and remodel RNA.

Nucleic Acids Res 2019 02;47(4):2130-2142

Department of Biochemistry, Duke University School of Medicine, Durham, NC 27710, USA.

Kinetoplastid RNA (kRNA) editing takes place in the mitochondria of kinetoplastid protists and creates translatable mRNAs by uridine insertion/deletion. Extensively edited (pan-edited) transcripts contain quadruplex forming guanine stretches, which must be remodeled to promote uridine insertion/deletion. Here we show that the RRM domain of the essential kRNA-editing factor TbRGG2 binds poly(G) and poly(U) RNA and can unfold both. A region C-terminal to the RRM mediates TbRGG2 dimerization, enhancing RNA binding. A RRM-U4 RNA structure reveals a unique RNA-binding mechanism in which the two RRMs of the dimer employ aromatic residues outside the canonical RRM RNA-binding motifs to encase and wrench open the RNA, while backbone atoms specify the uridine bases. Notably, poly(G) RNA is bound via a different binding surface. Thus, these data indicate that TbRGG2 RRM can bind and remodel several RNA substrates suggesting how it might play multiple roles in the kRNA editing process.
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http://dx.doi.org/10.1093/nar/gky1259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6393287PMC
February 2019

A Bacterial Chromosome Structuring Protein Binds Overtwisted DNA to Stimulate Type II Topoisomerases and Enable DNA Replication.

Cell 2018 10 13;175(2):583-597.e23. Epub 2018 Sep 13.

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address:

When DNA is unwound during replication, it becomes overtwisted and forms positive supercoils in front of the translocating DNA polymerase. Unless removed or dissipated, this superhelical tension can impede replication elongation. Topoisomerases, including gyrase and topoisomerase IV in bacteria, are required to relax positive supercoils ahead of DNA polymerase but may not be sufficient for replication. Here, we find that GapR, a chromosome structuring protein in Caulobacter crescentus, is required to complete DNA replication. GapR associates in vivo with positively supercoiled chromosomal DNA, and our biochemical and structural studies demonstrate that GapR forms a dimer-of-dimers that fully encircles overtwisted DNA. Further, we show that GapR stimulates gyrase and topo IV to relax positive supercoils, thereby enabling DNA replication. Analogous chromosome structuring proteins that locate to the overtwisted DNA in front of replication forks may be present in other organisms, similarly helping to recruit and stimulate topoisomerases during DNA replication.
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http://dx.doi.org/10.1016/j.cell.2018.08.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6173638PMC
October 2018

A potentially abundant junctional RNA motif stabilized by mA and Mg.

Nat Commun 2018 07 17;9(1):2761. Epub 2018 Jul 17.

Department of Biochemistry, Duke University School of Medicine, Durham, NC, 27710, USA.

N-Methyladenosine (mA) is an abundant post-transcriptional RNA modification that influences multiple aspects of gene expression. In addition to recruiting proteins, mA can modulate RNA function by destabilizing base pairing. Here, we show that when neighbored by a 5' bulge, mA stabilizes mA-U base pairs, and global RNA structure by ~1 kcal mol. The bulge most likely provides the flexibility needed to allow optimal stacking between the methyl group and 3' neighbor through a conformation that is stabilized by Mg. A bias toward this motif can help explain the global impact of methylation on RNA structure in transcriptome-wide studies. While mA embedded in duplex RNA is poorly recognized by the YTH domain reader protein and mA antibodies, both readily recognize mA in this newly identified motif. The results uncover potentially abundant and functional mA motifs that can modulate the epitranscriptomic structure landscape with important implications for the interpretation of transcriptome-wide data.
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http://dx.doi.org/10.1038/s41467-018-05243-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6050335PMC
July 2018

The crystal structure of the RsbN-σBldN complex from Streptomyces venezuelae defines a new structural class of anti-σ factor.

Nucleic Acids Res 2018 08;46(14):7405-7417

Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.

Streptomyces are filamentous bacteria with a complex developmental life cycle characterized by the formation of spore-forming aerial hyphae. Transcription of the chaplin and rodlin genes, which are essential for aerial hyphae production, is directed by the extracytoplasmic function (ECF) σ factor BldN, which is in turn controlled by an anti-σ factor, RsbN. RsbN shows no sequence similarity to known anti-σ factors and binds and inhibits BldN in an unknown manner. Here we describe the 2.23 Å structure of the RsbN-BldN complex. The structure shows that BldN harbors σ2 and σ4 domains that are individually similar to other ECF σ domains, which bind -10 and -35 promoter regions, respectively. The anti-σ RsbN consists of three helices, with α3 forming a long helix embraced between BldN σ2 and σ4 while RsbN α1-α2 dock against σ4 in a manner that would block -35 DNA binding. RsbN binding also freezes BldN in a conformation inactive for simultaneous -10 and -35 promoter interaction and RNAP binding. Strikingly, RsbN is structurally distinct from previously solved anti-σ proteins. Thus, these data characterize the molecular determinants controlling a central Streptomyces developmental switch and reveal RsbN to be the founding member of a new structural class of anti-σ factor.
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http://dx.doi.org/10.1093/nar/gky493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6101532PMC
August 2018

Structural basis of O-GlcNAc recognition by mammalian 14-3-3 proteins.

Proc Natl Acad Sci U S A 2018 06 21;115(23):5956-5961. Epub 2018 May 21.

Department of Biochemistry, Duke University School of Medicine, Durham, NC 27710;

O-GlcNAc is an intracellular posttranslational modification that governs myriad cell biological processes and is dysregulated in human diseases. Despite this broad pathophysiological significance, the biochemical effects of most O-GlcNAcylation events remain uncharacterized. One prevalent hypothesis is that O-GlcNAc moieties may be recognized by "reader" proteins to effect downstream signaling. However, no general O-GlcNAc readers have been identified, leaving a considerable gap in the field. To elucidate O-GlcNAc signaling mechanisms, we devised a biochemical screen for candidate O-GlcNAc reader proteins. We identified several human proteins, including 14-3-3 isoforms, that bind O-GlcNAc directly and selectively. We demonstrate that 14-3-3 proteins bind O-GlcNAc moieties in human cells, and we present the structures of 14-3-3β/α and γ bound to glycopeptides, providing biophysical insights into O-GlcNAc-mediated protein-protein interactions. Because 14-3-3 proteins also bind to phospho-serine and phospho-threonine, they may integrate information from O-GlcNAc and O-phosphate signaling pathways to regulate numerous physiological functions.
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http://dx.doi.org/10.1073/pnas.1722437115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6003352PMC
June 2018

The MerR-like protein BldC binds DNA direct repeats as cooperative multimers to regulate Streptomyces development.

Nat Commun 2018 03 19;9(1):1139. Epub 2018 Mar 19.

Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK.

Streptomycetes are notable for their complex life cycle and production of most clinically important antibiotics. A key factor that controls entry into development and the onset of antibiotic production is the 68-residue protein, BldC. BldC is a putative DNA-binding protein related to MerR regulators, but lacks coiled-coil dimerization and effector-binding domains characteristic of classical MerR proteins. Hence, the molecular function of the protein has been unclear. Here we show that BldC is indeed a DNA-binding protein and controls a regulon that includes other key developmental regulators. Intriguingly, BldC DNA-binding sites vary significantly in length. Our BldC-DNA structures explain this DNA-binding capability by revealing that BldC utilizes a DNA-binding mode distinct from MerR and other known regulators, involving asymmetric head-to-tail oligomerization on DNA direct repeats that results in dramatic DNA distortion. Notably, BldC-like proteins radiate throughout eubacteria, establishing BldC as the founding member of a new structural family of regulators.
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http://dx.doi.org/10.1038/s41467-018-03576-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5859096PMC
March 2018

Dissection of the molecular circuitry controlling virulence in .

Genes Dev 2017 08 1;31(15):1549-1560. Epub 2017 Sep 1.

Department of Biochemistry, Duke University School of Medicine, Durham, North Carolina 27710, USA.

the etiological agent of tularemia, is one of the most infectious bacteria known. Because of its extreme pathogenicity, is classified as a category A bioweapon by the US government. virulence stems from genes encoded on the pathogenicity island (FPI). An unusual set of regulators-the heteromeric macrophage growth locus protein A (MglA)-stringent starvation protein A (SspA) complex and the DNA-binding protein pathogenicity island gene regulator (PigR)-activates FPI transcription and thus is essential for virulence. Intriguingly, the second messenger, guanosine-tetraphosphate (ppGpp), which is produced during infection, is also involved in coordinating virulence; however, its role has been unclear. Here we identify MglA-SspA as a novel ppGpp-binding complex and describe structures of apo- and ppGpp-bound MglA-SspA. We demonstrate that MglA-SspA, which binds RNA polymerase (RNAP), also interacts with the C-terminal domain of PigR, thus anchoring the (MglA-SspA)-RNAP complex to the FPI promoter. Furthermore, we show that MglA-SspA must be bound to ppGpp to mediate high-affinity interactions with PigR. Thus, these studies unveil a novel pathway different from those described previously for regulation of transcription by ppGpp. The data also indicate that pathogenesis is controlled by a highly interconnected molecular circuitry in which the virulence machinery directly senses infection via a small molecule stress signal.
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http://dx.doi.org/10.1101/gad.303701.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5630020PMC
August 2017

Bacterial Nucleoid Occlusion: Multiple Mechanisms for Preventing Chromosome Bisection During Cell Division.

Subcell Biochem 2017;84:267-298

Department of Biochemistry, Duke University School of Medicine, 243 Nanaline H. Duke, Durham, NC, 27710, USA.

In most bacteria cell division is driven by the prokaryotic tubulin homolog, FtsZ, which forms the cytokinetic Z ring. Cell survival demands both the spatial and temporal accuracy of this process to ensure that equal progeny are produced with intact genomes. While mechanisms preventing septum formation at the cell poles have been known for decades, the means by which the bacterial nucleoid is spared from bisection during cell division, called nucleoid exclusion (NO), have only recently been deduced. The NO theory was originally posited decades ago based on the key observation that the cell division machinery appeared to be inhibited from forming near the bacterial nucleoid. However, what might drive the NO process was unclear. Within the last 10 years specific proteins have been identified as important mediators of NO. Arguably the best studied NO mechanisms are those employed by the Escherichia coli SlmA and Bacillus subtilis Noc proteins. Both proteins bind specific DNA sequences within selected chromosomal regions to act as timing devices. However, Noc and SlmA contain completely different structural folds and utilize distinct NO mechanisms. Recent studies have identified additional processes and factors that participate in preventing nucleoid septation during cell division. These combined data show multiple levels of redundancy as well as a striking diversity of mechanisms have evolved to protect cells against catastrophic bisection of the nucleoid. Here we discuss these recent findings with particular emphasis on what is known about the molecular underpinnings of specific NO machinery and processes.
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http://dx.doi.org/10.1007/978-3-319-53047-5_9DOI Listing
May 2019

The Streptomyces master regulator BldD binds c-di-GMP sequentially to create a functional BldD2-(c-di-GMP)4 complex.

Nucleic Acids Res 2017 Jun;45(11):6923-6933

Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, 10115 Berlin, Germany.

Streptomyces are ubiquitous soil bacteria that undergo a complex developmental transition coinciding with their production of antibiotics. This transition is controlled by binding of a novel tetrameric form of the second messenger, 3΄-5΄ cyclic diguanylic acid (c-di-GMP) to the master repressor, BldD. In all domains of life, nucleotide-based second messengers allow a rapid integration of external and internal signals into regulatory pathways that control cellular responses to changing conditions. c-di-GMP can assume alternative oligomeric states to effect different functions, binding to effector proteins as monomers, intercalated dimers or, uniquely in the case of BldD, as a tetramer. However, at physiological concentrations c-di-GMP is a monomer and little is known about how higher oligomeric complexes assemble on effector proteins and if intermediates in assembly pathways have regulatory significance. Here, we show that c-di-GMP binds BldD using an ordered, sequential mechanism and that BldD function necessitates the assembly of the BldD2-(c-di-GMP)4 complex.
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http://dx.doi.org/10.1093/nar/gkx287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499655PMC
June 2017

Structures of partition protein ParA with nonspecific DNA and ParB effector reveal molecular insights into principles governing Walker-box DNA segregation.

Genes Dev 2017 03 3;31(5):481-492. Epub 2017 Apr 3.

Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.

Walker-box partition systems are ubiquitous in nature and mediate the segregation of bacterial and archaeal DNA. Well-studied plasmid Walker-box partition modules require ParA, centromere-DNA, and a centromere-binding protein, ParB. In these systems, ParA-ATP binds nucleoid DNA and uses it as a substratum to deliver ParB-attached cargo DNA, and ParB drives ParA dynamics, allowing ParA progression along the nucleoid. How ParA-ATP binds nonspecific DNA and is regulated by ParB is unclear. Also under debate is whether ParA polymerizes on DNA to mediate segregation. Here we describe structures of key ParA segregation complexes. The ParA-β,γ-imidoadenosine 5'-triphosphate (AMPPNP)-DNA structure revealed no polymers. Instead, ParA-AMPPNP dimerization creates a multifaceted DNA-binding surface, allowing it to preferentially bind high-density DNA regions (HDRs). DNA-bound ParA-AMPPNP adopts a dimer conformation distinct from the ATP sandwich dimer, optimized for DNA association. Our ParA-AMPPNP-ParB structure reveals that ParB binds at the ParA dimer interface, stabilizing the ATPase-competent ATP sandwich dimer, ultimately driving ParA DNA dissociation. Thus, the data indicate how harnessing a conformationally adaptive dimer can drive large-scale cargo movement without the requirement for polymers and suggest a segregation mechanism by which ParA-ATP dimers equilibrate to HDRs shown to be localized near cell poles of dividing chromosomes, thus mediating equipartition of attached ParB-DNA substrates.
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http://dx.doi.org/10.1101/gad.296319.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5393062PMC
March 2017

Structure of the Z Ring-associated Protein, ZapD, Bound to the C-terminal Domain of the Tubulin-like Protein, FtsZ, Suggests Mechanism of Z Ring Stabilization through FtsZ Cross-linking.

J Biol Chem 2017 03 18;292(9):3740-3750. Epub 2017 Jan 18.

the Department of Biology, City College of City University of New York, New York, New York 10031, and

Cell division in most bacteria is mediated by the tubulin-like FtsZ protein, which polymerizes in a GTP-dependent manner to form the cytokinetic Z ring. A diverse repertoire of FtsZ-binding proteins affects FtsZ localization and polymerization to ensure correct Z ring formation. Many of these proteins bind the C-terminal domain (CTD) of FtsZ, which serves as a hub for FtsZ regulation. FtsZ ring-associated proteins, ZapA-D (Zaps), are important FtsZ regulatory proteins that stabilize FtsZ assembly and enhance Z ring formation by increasing lateral assembly of FtsZ protofilaments, which then form the Z ring. There are no structures of a Zap protein bound to FtsZ; therefore, how these proteins affect FtsZ polymerization has been unclear. Recent data showed ZapD binds specifically to the FtsZ CTD. Thus, to obtain insight into the ZapD-CTD interaction and how it may mediate FtsZ protofilament assembly, we determined the ZapD-FtsZ CTD structure to 2.67 Å resolution. The structure shows that the CTD docks within a hydrophobic cleft in the ZapD helical domain and adopts an unusual structure composed of two turns of helix separated by a proline kink. FtsZ CTD residue Phe-377 inserts into the ZapD pocket, anchoring the CTD in place and permitting hydrophobic contacts between FtsZ residues Ile-374, Pro-375, and Leu-378 with ZapD residues Leu-74, Trp-77, Leu-91, and Leu-174. The structural findings were supported by mutagenesis coupled with biochemical and studies. The combined data suggest that ZapD acts as a molecular cross-linking reagent between FtsZ protofilaments to enhance FtsZ assembly.
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http://dx.doi.org/10.1074/jbc.M116.773192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5339757PMC
March 2017

A three-dimensional ParF meshwork assembles through the nucleoid to mediate plasmid segregation.

Nucleic Acids Res 2017 04;45(6):3158-3171

Department of Biology, University of York, Wentworth Way, York YO10 5DD, UK.

Genome segregation is a fundamental step in the life cycle of every cell. Most bacteria rely on dedicated DNA partition proteins to actively segregate chromosomes and low copy-number plasmids. Here, by employing super resolution microscopy, we establish that the ParF DNA partition protein of the ParA family assembles into a three-dimensional meshwork that uses the nucleoid as a scaffold and periodically shuttles between its poles. Whereas ParF specifies the territory for plasmid trafficking, the ParG partner protein dictates the tempo of ParF assembly cycles and plasmid segregation events by stimulating ParF adenosine triphosphate hydrolysis. Mutants in which this ParG temporal regulation is ablated show partition deficient phenotypes as a result of either altered ParF structure or dynamics and indicate that ParF nucleoid localization and dynamic relocation, although necessary, are not sufficient per se to ensure plasmid segregation. We propose a Venus flytrap model that merges the concepts of ParA polymerization and gradient formation and speculate that a transient, dynamic network of intersecting polymers that branches into the nucleoid interior is a widespread mechanism to distribute sizeable cargos within prokaryotic cells.
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http://dx.doi.org/10.1093/nar/gkw1302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389482PMC
April 2017

Structures of the activator of K. pneumonia biofilm formation, MrkH, indicates PilZ domains involved in c-di-GMP and DNA binding.

Proc Natl Acad Sci U S A 2016 09 22;113(36):10067-72. Epub 2016 Aug 22.

Department of Biochemistry, Duke University School of Medicine, Durham, NC 27710.

The pathogenesis of Klebsiella pneumonia is linked to the bacteria's ability to form biofilms. Mannose-resistant Klebsiella-like (Mrk) hemagglutinins are critical for K pneumonia biofilm development, and the expression of the genes encoding these proteins is activated by a 3',5'-cyclic diguanylic acid (c-di-GMP)-regulated transcription factor, MrkH. To gain insight into MrkH function, we performed structural and biochemical analyses. Data revealed MrkH to be a monomer with a two-domain architecture consisting of a PilZ C-domain connected to an N domain that unexpectedly also harbors a PilZ-like fold. Comparison of apo- and c-di-GMP-bound MrkH structures reveals a large 138° interdomain rotation that is induced by binding an intercalated c-di-GMP dimer. c-di-GMP interacts with PilZ C-domain motifs 1 and 2 (RxxxR and D/NxSxxG) and a newly described c-di-GMP-binding motif in the MrkH N domain. Strikingly, these c-di-GMP-binding motifs also stabilize an open state conformation in apo MrkH via contacts from the PilZ motif 1 to residues in the C-domain motif 2 and the c-di-GMP-binding N-domain motif. Use of the same regions in apo structure stabilization and c-di-GMP interaction allows distinction between the states. Indeed, domain reorientation by c-di-GMP complexation with MrkH, which leads to a highly compacted structure, suggests a mechanism by which the protein is activated to bind DNA. To our knowledge, MrkH represents the first instance of specific DNA binding mediated by PilZ domains. The MrkH structures also pave the way for the rational design of inhibitors that target K pneumonia biofilm formation.
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http://dx.doi.org/10.1073/pnas.1607503113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5018759PMC
September 2016

Structures of trehalose-6-phosphate phosphatase from pathogenic fungi reveal the mechanisms of substrate recognition and catalysis.

Proc Natl Acad Sci U S A 2016 06 15;113(26):7148-53. Epub 2016 Jun 15.

Department of Biochemistry, Duke University School of Medicine, Durham, NC 27710;

Trehalose is a disaccharide essential for the survival and virulence of pathogenic fungi. The biosynthesis of trehalose requires trehalose-6-phosphate synthase, Tps1, and trehalose-6-phosphate phosphatase, Tps2. Here, we report the structures of the N-terminal domain of Tps2 (Tps2NTD) from Candida albicans, a transition-state complex of the Tps2 C-terminal trehalose-6-phosphate phosphatase domain (Tps2PD) bound to BeF3 and trehalose, and catalytically dead Tps2PD(D24N) from Cryptococcus neoformans bound to trehalose-6-phosphate (T6P). The Tps2NTD closely resembles the structure of Tps1 but lacks any catalytic activity. The Tps2PD-BeF3-trehalose and Tps2PD(D24N)-T6P complex structures reveal a "closed" conformation that is effected by extensive interactions between each trehalose hydroxyl group and residues of the cap and core domains of the protein, thereby providing exquisite substrate specificity. Disruption of any of the direct substrate-protein residue interactions leads to significant or complete loss of phosphatase activity. Notably, the Tps2PD-BeF3-trehalose complex structure captures an aspartyl-BeF3 covalent adduct, which closely mimics the proposed aspartyl-phosphate intermediate of the phosphatase catalytic cycle. Structures of substrate-free Tps2PD reveal an "open" conformation whereby the cap and core domains separate and visualize the striking conformational changes effected by substrate binding and product release and the role of two hinge regions centered at approximately residues 102-103 and 184-188. Significantly, tps2Δ, tps2NTDΔ, and tps2D705N strains are unable to grow at elevated temperatures. Combined, these studies provide a deeper understanding of the substrate recognition and catalytic mechanism of Tps2 and provide a structural basis for the future design of novel antifungal compounds against a target found in three major fungal pathogens.
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http://dx.doi.org/10.1073/pnas.1601774113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4932955PMC
June 2016

Structures of Pathogenic Fungal FKBP12s Reveal Possible Self-Catalysis Function.

mBio 2016 Apr 26;7(2):e00492-16. Epub 2016 Apr 26.

Department of Biochemistry, Duke University School of Medicine, Durham, North Carolina, USA

Unlabelled: Invasive fungal infections remain difficult to treat and require novel targeting strategies. The 12-kDa FK506-binding protein (FKBP12) is a ubiquitously expressed peptidyl-prolyl isomerase with considerable homology between fungal pathogens and is thus a prime candidate for future targeting efforts to generate a panfungal strategy. Despite decades of research on FKBPs, their substrates and mechanisms of action remain unclear. Here we describe structural, biochemical, and in vivo analyses of FKBP12s from the pathogenic fungi Candida albicans, Candida glabrata, and Aspergillus fumigatus Strikingly, multiple apo A. fumigatus and C. albicans FKBP12 crystal structures revealed a symmetric, intermolecular interaction involving the deep insertion of an active-site loop proline into the active-site pocket of an adjacent subunit. Such interactions have not been observed in previous FKBP structures. This finding indicates the possibility that this is a self-substrate interaction unique to the A. fumigatus and C. albicans fungal proteins that contain this central proline. Structures obtained with the proline in the cis and trans states provide more data in support of self-catalysis. Moreover, cysteine cross-linking experiments captured the interacting dimer, supporting the idea that it forms in solution. Finally, genetic studies exploring the impact of mutations altering the central proline and an adjacent residue provide evidence that any dimeric state formed in vivo, where FKBP12 concentrations are low, is transient. Taken together, these findings suggest a unique mechanism of self-substrate regulation by fungal FKBP12s, lending further novel understanding of this protein for future drug-targeting efforts.

Importance: FKBP12 is a cis-trans peptidyl-prolyl isomerase that plays key roles in cellular protein homeostasis. FKBP12s also bind the immunosuppressive drug FK506 to inhibit the phosphatase calcineurin (CaN). CaN is required for virulence of A. fumigatus, C. albicans, C. glabrata, and other deadly fungal pathogens, marking FKBP12 and CaN as potential broad-spectrum drug targets. Here we describe structures of fungal FKBP12s. Multiple apo A. fumigatus and C. albicans FKBP12 structures reveal the insertion of a proline, conspicuously conserved in these proteins, into the active sites of adjacent molecules. This suggests that these proteins might serve as their own substrates. Cysteine disulfide trapping experiments provide support for this self-interaction and hence possible intermolecular catalysis by these enzymes.
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http://dx.doi.org/10.1128/mBio.00492-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4850266PMC
April 2016

Structures of the nucleoid occlusion protein SlmA bound to DNA and the C-terminal domain of the cytoskeletal protein FtsZ.

Proc Natl Acad Sci U S A 2016 May 18;113(18):4988-93. Epub 2016 Apr 18.

Department of Biochemistry, Duke University School of Medicine, Durham, NC 27710.

Cell division in most prokaryotes is mediated by FtsZ, which polymerizes to create the cytokinetic Z ring. Multiple FtsZ-binding proteins regulate FtsZ polymerization to ensure the proper spatiotemporal formation of the Z ring at the division site. The DNA-binding protein SlmA binds to FtsZ and prevents Z-ring formation through the nucleoid in a process called "nucleoid occlusion" (NO). As do most FtsZ-accessory proteins, SlmA interacts with the conserved C-terminal domain (CTD) that is connected to the FtsZ core by a long, flexible linker. However, SlmA is distinct from other regulatory factors in that it must be DNA-bound to interact with the FtsZ CTD. Few structures of FtsZ regulator-CTD complexes are available, but all reveal the CTD bound as a helix. To deduce the molecular basis for the unique SlmA-DNA-FtsZ CTD regulatory interaction and provide insight into FtsZ-regulator protein complex formation, we determined structures of Escherichia coli, Vibrio cholera, and Klebsiella pneumonia SlmA-DNA-FtsZ CTD ternary complexes. Strikingly, the FtsZ CTD does not interact with SlmA as a helix but binds as an extended conformation in a narrow, surface-exposed pocket formed only in the DNA-bound state of SlmA and located at the junction between the DNA-binding and C-terminal dimer domains. Binding studies are consistent with the structure and underscore key interactions in complex formation. Combined, these data reveal the molecular basis for the SlmA-DNA-FtsZ interaction with implications for SlmA's NO function and underscore the ability of the FtsZ CTD to adopt a wide range of conformations, explaining its ability to bind diverse regulatory proteins.
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http://dx.doi.org/10.1073/pnas.1602327113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4983851PMC
May 2016

Molecular insights into DNA binding and anchoring by the Bacillus subtilis sporulation kinetochore-like RacA protein.

Nucleic Acids Res 2016 06 16;44(11):5438-49. Epub 2016 Apr 16.

Department of Biochemistry, Duke University School of Medicine, 255 Nanaline H. Duke, Durham, NC 27710, USA.

During Bacillus subtilis sporulation, segregating sister chromosomes are anchored to cell poles and the chromosome is remodeled into an elongated structure called the axial filament. Data indicate that a developmentally regulated protein called RacA is involved in these functions. To gain insight into how RacA performs these diverse processes we performed a battery of structural and biochemical analyses. These studies show that RacA contains an N-terminal winged-helix-turn-helix module connected by a disordered region to a predicted coiled-coil domain. Structures capture RacA binding the DNA using distinct protein-protein interfaces and employing adjustable DNA docking modes. This unique DNA binding mechanism indicates how RacA can both specifically recognize its GC-rich centromere and also non-specifically bind the DNA. Adjacent RacA molecules within the protein-DNA structure interact leading to DNA compaction, suggesting a mechanism for axial filament formation. We also show that the RacA C-domain coiled coil directly contacts the coiled coil region of the polar protein DivIVA, which anchors RacA and hence the chromosome to the pole. Thus, our combined data reveal unique DNA binding properties by RacA and provide insight into the DNA remodeling and polar anchorage functions of the protein.
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http://dx.doi.org/10.1093/nar/gkw248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4914108PMC
June 2016

Structural and Functional Analyses Reveal Insights into the Molecular Properties of the Escherichia coli Z Ring Stabilizing Protein, ZapC.

J Biol Chem 2016 Jan 10;291(5):2485-98. Epub 2015 Dec 10.

Department of Biology, The City College of CUNY, New York, New York 10031, and The Graduate Center of CUNY, New York, New York 10016

In Escherichia coli cell division is driven by the tubulin-like GTPase, FtsZ, which forms the cytokinetic Z-ring. The Z-ring serves as a dynamic platform for the assembly of the multiprotein divisome, which catalyzes membrane cleavage to create equal daughter cells. Several proteins effect FtsZ assembly, thereby providing spatiotemporal control over cell division. One important class of FtsZ interacting/regulatory proteins is the Z-ring-associated proteins, Zaps, which typically modulate Z-ring formation by increasing lateral interactions between FtsZ protofilaments. Strikingly, these Zap proteins show no discernable sequence similarity, suggesting that they likely harbor distinct structures and mechanisms. The 19.8-kDa ZapC in particular shows no homology to any known protein. To gain insight into ZapC function, we determined its structure to 2.15 Å and performed genetic and biochemical studies. ZapC is a monomer composed of two domains, an N-terminal α/β region and a C-terminal twisted β barrel-like domain. The structure contains two pockets, one on each domain. The N-domain pocket is lined with residues previously implicated to be important for ZapC function as an FtsZ bundler. The adjacent C-domain pocket contains a hydrophobic center surrounded by conserved basic residues. Mutagenesis analyses indicate that this pocket is critical for FtsZ binding. An extensive FtsZ binding surface is consistent with the fact that, unlike many FtsZ regulators, ZapC binds the large FtsZ globular core rather than C-terminal tail, and the presence of two adjacent pockets suggests possible mechanisms for ZapC-mediated FtsZ bundling.
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http://dx.doi.org/10.1074/jbc.M115.697037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4732229PMC
January 2016
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