Publication date: 14 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 16

Author(s): Mauricio H. Pontes, Anastasia Sevostyanova, Eduardo A. Groisman

Adenosine triphosphate (ATP) is the energy currency of living cells. Even though ATP powers virtually all energy-dependent activities, most cellular ATP is utilized in protein synthesis via tRNA aminoacylation and guanosine triphosphate regeneration. Magnesium (Mg2+), the most common divalent cation in living cells, plays crucial roles in protein synthesis by maintaining the structure of ribosomes, participating in the biochemistry of translation initiation and functioning as a counterion for ATP. A non-physiological increase in ATP levels hinders growth in cells experiencing Mg2+ limitation because ATP is the most abundant nucleotide triphosphate in the cell, and Mg2+ is also required for the stabilization of the cytoplasmic membrane and as a cofactor for essential enzymes. We propose that organisms cope with Mg2+ limitation by decreasing ATP levels and ribosome production, thereby reallocating Mg2+ to indispensable cellular processes.
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Publication date: 14 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 16

Author(s): Sandra G. Schrempp, Martin van der Laan

Publication date: 14 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 16

Author(s): Jarungjit Rujiviphat, Michael K. Wong, Amy Won, Yu-ling Shih, Christopher M. Yip, G. Angus McQuibban

Large GTPases of the dynamin superfamily promote membrane fusion and division, processes that are crucial for intracellular trafficking and organellar dynamics. To promote membrane scission, dynamin proteins polymerize, wrap around, and constrict the membrane; however, the mechanism underlying their role in membrane fusion remains unclear. We previously reported that the mitochondrial dynamin-related protein mitochondrial genome maintenance 1 (Mgm1) mediates fusion by first tethering opposing membranes and then undergoing a nucleotide-dependent structural transition. However, it is still unclear how Mgm1 directly affects the membrane to drive fusion of tethered membranes. Here, we show that Mgm1 association with the membrane alters the topography of the membrane, promoting local membrane bending. We also demonstrate that Mgm1 creates membrane ruffles resulting in the formation of tubular structures on both supported lipid bilayers and liposomes. These data suggest that Mgm1 membrane interactions impose a mechanical force on the membrane to overcome the hydrophilic repulsion of the phospholipid head groups and initiate the fusion reaction. The work reported here provides new insights into a possible mechanism of Mgm1-driven mitochondrial membrane fusion and sheds light into how members of the dynamin superfamily function as fusion molecules.
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Publication date: 14 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 16

Author(s): Andrew Routh, Steven R. Head, Phillip Ordoukhanian, John E. Johnson

We present a simple method called “ClickSeq” for NGS (next-generation sequencing) library synthesis that uses click chemistry rather than enzymatic reactions for the ligation of Illumina sequencing adaptors. In ClickSeq, randomly primed reverse transcription reactions are supplemented with azido-2′,3′-dideoxynucleotides that randomly terminate DNA synthesis and release 3′-azido-blocked cDNA fragments in a process akin to dideoxy-Sanger sequencing. Purified fragments are “click ligated” via copper-catalyzed alkyne-azide cycloaddition to DNA oligos modified with a 5′-alkyne group. This generates ssDNA molecules containing an unnatural triazole-linked DNA backbone that is sufficiently biocompatible for PCR amplification to generate a cDNA library for RNAseq. Here, we analyze viral RNAs and mRNA to demonstrate that ClickSeq produces unbiased NGS libraries with low error rates comparable to standard methods. Importantly, ClickSeq is robust against common artifacts of NGS such as chimera formation and artifactual recombination with fewer than 3 aberrant events detected per million reads.
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Publication date: 14 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 16

Author(s): Leopold Kong, Rameshwar U. Kadam, Erick Giang, Tinashe B. Ruwona, Travis Nieusma, Jeffrey C. Culhane, Robyn L. Stanfield, Philip E. Dawson, Ian A. Wilson, Mansun Law

Hepatitis C virus (HCV) is a positive-strand RNA virus within the Flaviviridae family. The viral “spike” of HCV is formed by two envelope glycoproteins, E1 and E2, which together mediate viral entry by engaging host receptors and undergoing conformational changes to facilitate membrane fusion. While E2 can be readily produced in the absence of E1, E1 cannot be expressed without E2 and few reagents, including monoclonal antibodies (mAbs), are available for study of this essential HCV glycoprotein. A human mAb to E1, IGH526, was previously reported to cross-neutralize different HCV isolates, and therefore, we sought to further characterize the IGH526 neutralizing epitope to obtain information for vaccine design. We found that mAb IGH526 bound to a discontinuous epitope, but with a major component corresponding to E1 residues 314–324. The crystal structure of IGH526 Fab with this E1 glycopeptide at 1.75Å resolution revealed that the antibody binds to one face of an α-helical peptide. Single mutations on the helix substantially lowered IGH526 binding but did not affect neutralization, indicating either that multiple mutations are required or that additional regions are recognized by the antibody in the context of the membrane-associated envelope oligomer. Molecular dynamics simulations indicate that the free peptide is flexible in solution, suggesting that it requires stabilization for use as a candidate vaccine immunogen.
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Publication date: 14 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 16

Author(s): Ananthamohan Kalyani, Parshuram J. Sonawane, Abrar Ali Khan, Lakshmi Subramanian, Georg B. Ehret, Ajit S. Mullasari, Nitish R. Mahapatra

Renalase, a recently identified oxidoreductase, is emerging as a novel regulator of cardiovascular and metabolic disease states. The mechanism of regulation of renalase gene, especially at the post-transcriptional level, is completely unknown. We set out to investigate the possible role of microRNAs in regulation of renalase gene in this study. Computational predictions using multiple algorithms coupled with systematic functional analysis revealed specific interactions of miR-29a/b/c and miR-146a/b with mouse and human renalase 3′-UTR (untranslated region) in cultured cells. Next, we estimated miR-29b and miR-146a, as well as renalase expression, in genetically hypertensive blood pressure high and genetically hypotensive blood pressure low mice. Kidney tissues from blood pressure high mice showed diminished (~1.6- to 1.8-fold) renalase mRNA/protein levels and elevated (~2.2-fold) miR-29b levels as compared to blood pressure low mice. A common single nucleotide polymorphism in human renalase 3′-UTR (C/T; rs10749571) creates a binding site for miR-146a; consistently, miR-146a down-regulated human renalase 3′-UTR/luciferase activity in case of the T allele suggesting its potential role in regulation of renalase in humans. Indeed, genome-wide association studies revealed directionally concordant association of rs10749571 with diastolic blood pressure, glucose and triglyceride levels in large human populations (n ≈58,000–96,000 subjects). This study provides evidence for post-transcriptional regulation of renalase gene by miR-29 and miR-146 and has implications for inter-individual variations on cardiometabolic traits.
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Publication date: 14 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 16

Author(s): Jérémy Loyau, Gérard Didelot, Pauline Malinge, Ulla Ravn, Giovanni Magistrelli, Jean-François Depoisier, Guillemette Pontini, Yves Poitevin, Marie Kosco-Vilbois, Nicolas Fischer, Stéphane Thore, François Rousseau

Hu 15C1 is a potent anti-human Toll-like receptor 4 (TLR4) neutralizing antibody. To better understand the molecular basis of its biological activity, we used a multidisciplinary approach to generate an accurate model of the Hu 15C1–TLR4 complex. By combining site-directed mutagenesis, in vitro antibody evolution, affinity measurements and X-ray crystallography of Fab fragments, we identified key interactions across the Hu 15C1–TLR4 interface. These contact points were used as restraints to predict the structure of the Fab region of Hu 15C1 bound to TLR4 using computational molecular docking. This model was further evaluated and validated by additional site-directed mutagenesis studies. The predicted structure of the Hu 15C1–TLR4 complex indicates that the antibody antagonizes the receptor dimerization necessary for its activation. This study exemplifies how iterative cycles of antibody engineering can facilitate the discovery of components of antibody-target interactions.
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Publication date: 14 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 16

Author(s): Agato Murata, Yuji Ito, Risa Kashima, Saori Kanbayashi, Kei Nanatani, Chihiro Igarashi, Masaki Okumura, Kenji Inaba, Takashi Tokino, Satoshi Takahashi, Kiyoto Kamagata

One-dimensional (1D) sliding of the tumor suppressor p53 along DNA is an essential dynamics required for its efficient search for the binding sites in the genome. To address how the search process of p53 is affected by the changes in the concentration of Mg2+ and Ca2+ after the cell damages, we investigated its sliding dynamics at different concentrations of the divalent cations. The 1D sliding trajectories of p53 along the stretched DNA were measured by using single-molecule fluorescence microscopy. The averaged diffusion coefficient calculated from the mean square displacement of p53 on DNA increased significantly at the higher concentration of Mg2+ or Ca2+, indicating that the divalent cations accelerate the sliding likely by weakening the DNA–p53 interaction. In addition, two distributions were identified in the displacement of the observed trajectories of p53, demonstrating the presence of the fast and slow sliding modes having large and small diffusion coefficients, respectively. A coreless mutant of p53, in which the core domain was deleted, showed only a single mode whose diffusion coefficient is about twice that of the fast mode for the full-length p53. Thus, the two modes are likely the result of the tight and loose interactions between the core domain of p53 and DNA. These results demonstrated clearly that the 1D sliding dynamics of p53 is strongly dependent on the concentration of Mg2+ and Ca2+, which maintains the search distance of p53 along DNA in cells that lost homeostatic control of the divalent cations.
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Publication date: 14 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 16

Author(s): Olga Martzoukou, Mayia Karachaliou, Vassilis Yalelis, James Leung, Bernadette Byrne, Sotiris Amillis, George Diallinas

Central to the process of transmembrane cargo trafficking is the successful folding and exit from the ER (endoplasmic reticulum) through packaging in COPII vesicles. Here, we use the UapA purine transporter of Aspergillus nidulans to investigate the role of cargo oligomerization in membrane trafficking. We show that UapA oligomerizes (at least dimerizes) and that oligomerization persists upon UapA endocytosis and vacuolar sorting. Using a validated bimolecular fluorescence complementation assay, we provide evidence that a UapA oligomerization is associated with ER-exit and turnover, as ER-retained mutants due to either modification of a Tyr-based N-terminal motif or partial misfolding physically associate but do not associate properly. Co-expression of ER-retained mutants with wild-type UapA leads to in trans plasma membrane localization of the former, confirming that oligomerization initiates in the ER. Genetic suppression of an N-terminal mutation in the Tyr motif and mutational analysis suggest that transmembrane α-helix 7 affects the oligomerization interface. Our results reveal that transporter oligomerization is essential for membrane trafficking and turnover and is a common theme in fungi and mammalian cells.
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Publication date: 14 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 16

Author(s): Yun Mou, Po-Ssu Huang, Leonard M. Thomas, Stephen L. Mayo

In standard implementations of computational protein design, a positive-design approach is used to predict sequences that will be stable on a given backbone structure. Possible competing states are typically not considered, primarily because appropriate structural models are not available. One potential competing state, the domain-swapped dimer, is especially compelling because it is often nearly identical with its monomeric counterpart, differing by just a few mutations in a hinge region. Molecular dynamics (MD) simulations provide a computational method to sample different conformational states of a structure. Here, we tested whether MD simulations could be used as a post-design screening tool to identify sequence mutations leading to domain-swapped dimers. We hypothesized that a successful computationally designed sequence would have backbone structure and dynamics characteristics similar to that of the input structure and that, in contrast, domain-swapped dimers would exhibit increased backbone flexibility and/or altered structure in the hinge-loop region to accommodate the large conformational change required for domain swapping. While attempting to engineer a homodimer from a 51-amino-acid fragment of the monomeric protein engrailed homeodomain (ENH), we had instead generated a domain-swapped dimer (ENH_DsD). MD simulations on these proteins showed increased B-factors derived from MD simulation in the hinge loop of the ENH_DsD domain-swapped dimer relative to monomeric ENH. Two point mutants of ENH_DsD designed to recover the monomeric fold were then tested with an MD simulation protocol. The MD simulations suggested that one of these mutants would adopt the target monomeric structure, which was subsequently confirmed by X-ray crystallography.
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Publication date: 14 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 16

Author(s): Somnath Mukherjee, Marcin Ura, Robert J. Hoey, Anthony A. Kossiakoff

Reversible, high-affinity immobilization tags are critical tools for myriad biological applications. However, inherent issues are associated with a number of the current methods of immobilization. Particularly, a critical element in phage display sorting is functional immobilization of target proteins. To circumvent these problems, we have used a mutant (N5A) of calmodulin binding peptide (CBP) as an immobilization tag in phage display sorting. The immobilization relies on the ultra high affinity of calmodulin to N5A mutant CBP (RWKKNFIAVSAANRFKKIS) in presence of calcium (K D ~2pM), which can be reversed by EDTA allowing controlled “capture and release” of the specific binders. To evaluate the capabilities of this system, we chose eight targets, some of which were difficult to overexpress and purify with other tags and some had failed in sorting experiments. In all cases, specific binders were generated using a Fab phage display library with CBP-fused constructs. K D values of the Fabs were in subnanomolar to low nanomolar (nM) ranges and were successfully used to selectively recognize antigens in cell-based experiments. Some of these targets were problematic even without any tag; thus, the fact that all led to successful selection endpoints means that borderline cases can be worked on with a high probability of a positive outcome. Taken together with examples of successful case specific, high-level applications like generation of conformation-, epitope- and domain-specific Fabs, we feel that the CBP tag embodies all the attributes of covalent immobilization tags but does not suffer from some of their well-documented drawbacks.
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Publication date: 14 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 16

Author(s): Derya Meral, Brigita Urbanc

Publication date: Available online 14 August 2015
Source:Journal of Molecular Biology

Author(s): Sachini U. Siriwardena, Thisari A. Guruge, Ashok S. Bhagwat

Human APOBEC3B deaminates cytosines in DNA and belongs to the AID/APOBEC family of enzymes. These proteins are involved in innate and adaptive immunity and may cause mutations in a variety of cancers. To characterize its ability to convert cytosines into uracils, we tested several derivatives of APOBEC3B gene for their ability to cause mutations in Escherichia coli. Through this analysis, a methionine residue at the junction of the amino-terminal domain (NTD) and the carboxy-terminal domain (CTD) was found to be essential for high mutagenicity. Properties of mutants with substitutions at this position, examination of existing molecular structures of APOBEC3 family members and molecular modeling suggest that this residue is essential for the structural stability of this family of proteins. The APOBEC3B CTD with the highest mutational activity was purified to homogeneity and its kinetic parameters were determined. Size-exclusion chromatography of the CTD monomer showed that it is in equilibrium with its dimeric form and matrix-assisted laser desorption ionization time-of-flight analysis of the protein suggested that the dimer may be quite stable. The partially purified NTD did not show intrinsic deamination activity and did not enhance the activity of the CTD in biochemical assays. Finally, APOBEC3B was at least 10-fold less efficient at mutating 5-methylcytosine (5mC) to thymine than APOBEC3A in a genetic assay and was at least 10-fold less efficient at deaminating 5mC compared to C in biochemical assays. These results shed light on the structural organization of APOBEC3B catalytic domain, its substrate specificity and its possible role in causing genome-wide mutations.
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Publication date: Available online 14 August 2015
Source:Journal of Molecular Biology

Author(s): Hans Peter Sørensen, Peng Xu, Longguang Jiang, Tobias Kromann-Hansen, Knud J. Jensen, Mingdong Huang, Peter A. Andreasen

We have developed a new concept for designing peptidic protein modulators, by recombinantly fusing the peptidic modulator, with randomized residues, directly to the target protein via a linker and screening for internal modulation of the activity of the protein. We tested the feasibility of the concept by fusing a 10-residue-long, disulfide-bond-constrained inhibitory peptide, randomized in selected positions, to the catalytic domain of the serine protease murine urokinase-type plasminogen activator. High-affinity inhibitory peptide variants were identified as those that conferred to the fusion protease the lowest activity for substrate hydrolysis. The usefulness of the strategy was demonstrated by the selection of peptidic inhibitors of murine urokinase-type plasminogen activator with a low nanomolar affinity. The high affinity could not have been predicted by rational considerations, as the high affinity was associated with a loss of polar interactions and an increased binding entropy.
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Publication date: Available online 14 August 2015
Source:Journal of Molecular Biology

Author(s): Lipi Thukral, Simone Schwarze, Isabella Daidone, Hannes Neuweiler

Protein denatured states are the origin of both healthy and toxic conformational species. Denatured states of ultrafast folding proteins are of interest in mechanistic studies because they are energetically close to the kinetic bottleneck of folding. However, their transient nature makes them elusive to experiment. Here, we generated the denatured state of the helical domain BBL that is poised to fold in microseconds by a single-point mutation and combined circular dichroism spectroscopy, single-molecule fluorescence fluctuation analysis, and computer simulation to characterize its structure and dynamics. Circular dichroism showed a largely unfolded ensemble with marginal helix but significant β-sheet content. Main-chain structure and dynamics were unaffected by side-chain interactions that stabilize the native state, as revealed by site-directed mutagenesis and nanosecond loop closure kinetics probed by fluorescence correlation spectroscopy. Replica-exchange and constant-temperature molecular dynamics simulations showed a highly collapsed, hydrogen-bonded denatured state containing turn and β-sheet structure and few nucleating helices in an otherwise unfolded ensemble. An irregular β-hairpin element that connects helices in the native fold was poised to be formed. The surprising observation of β-structure in regions that form helices in the native state is reconciled by a generic low-energy pathway from the northwest quadrant of Ramachandran space to the helical basin present under folding conditions, proposed recently. Our results show that, indeed, rapid nucleation of helix emanates from β-structure formed early within a collapsed ensemble of unfolded conformers.
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Publication date: Available online 12 August 2015
Source:Journal of Molecular Biology

Author(s): Rasika M. Harshey, Jonathan D. Partridge

Flagella propel bacteria during both swimming and swarming, dispersing them widely. However, while swimming bacteria use chemotaxis to find nutrients and avoid toxic environments, swarming bacteria appear to suppress chemotaxis and to use the dynamics of their collective motion to continuously expand and acquire new territory, barrel through lethal chemicals in their path, carry along bacterial and fungal cargo that assists in exploration of new niches, and engage in group warfare for niche dominance. Here we focus on two aspects of swarming, which if understood, hold the promise of revealing new insights into microbial signaling and behavior, with ramifications beyond bacterial swarming. These are: how bacteria sense they are on a surface and turn on programs that promote movement, and how as dense packs they override scarcity and adversity.
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Publication date: Available online 12 August 2015
Source:Journal of Molecular Biology

Author(s): Frédérick Bouffard, Karine Plourde, Simon Bélanger, Geneviève Ouellette, Yvan Labrie, Francine Durocher

The FANC-BRCA DNA repair pathway is activated in response to interstrand crosslinks formed in DNA. A homozygous mutation in 1 of the 17 Fanconi anemia (FA) genes results in malfunctions of this pathway and development of FA syndrome. The integrity of this protein network is essential for good maintenance of DNA repair process and genome stability. Following the identification of an alternatively splice isoform of FANCE (Fanconi anemia complementation group E) significantly expressed in breast cancer individuals from high-risk non-BRCA1/2 families, we studied the impact of this FANCE splice isoform (FANCEΔ4) on DNA repair processes. We have demonstrated that FANCEΔ4 mRNA was efficiently translated into a functional protein and expressed in normal and breast cancer cell lines. Following treatment with the crosslinking agent mitomycin C, EUFA130 (FANCE-deficient) cells infected with FANCEΔ4 were blocked into G2/M phase, while cell survival was significantly reduced compared with FANCE-infected EUFA130 cells. In addition, FANCEΔ4 did not allow FANCD2 and FANCI monoubiquitination, which represents a crucial step of the FANC-BRCA functional pathway. As observed for FANCE wild-type protein, localization of FANCEΔ4 protein was confined to the nucleus following mitomycin C treatment. Although FANCEΔ4 protein showed interaction with FANCE, FANCEΔ4 did not support normal function of FANCE protein in this pathway and could have deleterious effects on FANCE protein activity. We have demonstrated that FANCEΔ4 seems to act as a regulator of FANCD2 protein expression level by promoting its degradation. This study highlights the importance of an efficient regulation of alternative splicing expression of FA genes for proper DNA repair.
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Publication date: Available online 8 August 2015
Source:Journal of Molecular Biology

Author(s): Philipp Trepte, Alexander Buntru, Konrad Klockmeier, Lindsay Willmore, Anup Arumughan, Christopher Secker, Martina Zenkner, Lydia Brusendorf, Kirstin Rau, Alexandra Redel, Erich E. Wanker

Mapping of protein-protein interactions (PPIs) is critical for understanding protein function and complex biological processes. Here, we present DULIP, a dual luminescence-based co-immunoprecipitation assay, for systematic PPI mapping in mammalian cells. DULIP is a second-generation luminescence-based PPI screening method for the systematic and quantitative analysis of co-immunoprecipitations using two different luciferase tags. Benchmarking studies with positive and negative PPI reference sets revealed that DULIP allows the detection of interactions with high sensitivity and specificity. Furthermore, the analysis of a PPI reference set with known binding affinities demonstrated that both low- and high-affinity interactions can be detected with DULIP assays. Finally, using the well-characterized interaction between Syntaxin-1 and Munc18, we found that DULIP is capable of detecting the effects of point mutations on interaction strength. Taken together, our studies demonstrate that DULIP is a sensitive and reliable method of great utility for systematic interactome research. It can be applied for interaction screening as well as for the validation of PPIs in mammalian cells. Moreover, DULIP permits the specific analysis of mutation-dependent binding patterns.
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Publication date: Available online 8 August 2015
Source:Journal of Molecular Biology

Author(s): Matthew Greetham, Emmanuel Skordalakes, David Lydall, Bernard A. Connolly

The telomere is present at the ends of all eukaryotic chromosomes and usually consists of repetitive TG-rich DNA that terminates in a single-stranded 3′ TG extension and a 5′ CA-rich recessed strand. A biochemical assay that allows the in vitro observation of exonuclease-catalyzed degradation (resection) of telomeres has been developed. The approach uses an oligodeoxynucleotide that folds to a stem–loop with a TG-rich double-stranded region and a 3′ single-stranded extension, typical of telomeres. Cdc13, the major component of the telomere-specific CST complex, strongly protects the recessed strand from the 5′→3′ exonuclease activity of the model exonuclease from bacteriophage λ. The isolated DNA binding domain of Cdc13 is less effective at shielding telomeres. Protection is specific, not being observed in control DNA lacking the specific TG-rich telomere sequence. RPA, the eukaryotic single-stranded DNA binding protein, also inhibits telomere resection. However, this protein is non-specific, equally hindering the degradation of non-telomere controls.
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Publication date: Available online 7 August 2015
Source:Journal of Molecular Biology

Author(s): Sukanya Chaudhury, Clarice de Azevedo Souza, Gregory V. Plano, Roberto N. De Guzman

The type III secretion system (T3SS) is essential in the pathogenesis of Yersinia pestis, the causative agent of plague. A small protein, LcrG, functions as a chaperone to the tip protein LcrV, and the LcrG–LcrV interaction is important in regulating protein secretion through the T3SS. The atomic structure of the LcrG family is currently unknown. However, because of its predicted helical propensity, many have suggested that the LcrG family forms a coiled-coil structure. Here, we show by NMR and CD spectroscopy that LcrG lacks a tertiary structure and it consists of three partially folded α-helices spanning residues 7–38, 41–46, and 58–73. NMR titrations of LcrG with LcrV show that the entire length of a truncated LcrG (residues 7–73) is involved in binding to LcrV. However, there is regional variation in how LcrG binds to LcrV. The C-terminal region of a truncated LcrG (residues 52–73) shows tight binding interaction with LcrV while the N-terminal region (residues 7–51) shows weaker interaction with LcrV. This suggests that there are at least two binding events when LcrG binds to LcrV. Biological assays and mutagenesis indicate that the C-terminal region of LcrG (residues 52–73) is important in blocking protein secretion through the T3SS. Our results reveal structural and mechanistic insights into the atomic conformation of LcrG and how it binds to LcrV.
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