A Hydrophobic Patch in the Competence-Stimulating Peptide, a Pneumococcal Competence Pheromone, Is Essential for Specificity and Biological Activity

ABSTRACT Induction of competence for natural genetic transformation in Streptococcus pneumoniae depends on pheromone-mediated cell-cell communication and a signaling pathway consisting of the competence-stimulating peptide (CSP), its membrane-embedded histidine kinase receptor ComD, and the cognate response regulator ComE. Extensive screening of pneumococcal isolates has revealed that two major CSP variants, CSP1 and CSP2, are found in members of this species. Even though the primary structures of CSP1 and CSP2 are about 50% identical, they are highly specific for their respective receptors, ComD1 and ComD2. In the present work, we have investigated the structural basis of this specificity by determining the three-dimensional structure of CSP1 from nuclear magnetic resonance data and comparing the agonist activity of a number of CSP1/CSP2 hybrid peptides toward the ComD1 and ComD2 receptors. Our results show that upon exposure to membrane-mimicking environments, the 17-amino-acid CSP1 pheromone adopts an amphiphilic α-helical configuration stretching from residue 6 to residue 12. Furthermore, the pattern of agonist activity displayed by the various hybrid peptides revealed that hydrophobic amino acids, some of which are situated on the nonpolar side of the α-helix, strongly contribute to CSP specificity. Together, these data indicate that the identified α-helix is an important structural feature of CSP1 which is essential for effective receptor recognition under natural conditions.

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TMEM106B, a risk factor for FTLD and aging, has an intrinsically disordered cytoplasmic domain

10.1101/317958 ◽

2018 ◽

Author(s):

Jian Kang ◽

Liangzhong Lim ◽

Jianxing Song

Keyword(s):

Risk Factor ◽

Three Dimensional ◽

Cytoplasmic Domain ◽

Dimensional Structure ◽

Structural Basis ◽

Biophysical Characterization ◽

General Role ◽

Intrinsically Disordered ◽

Autophagy Pathway ◽

Α Helix

AbstractTMEM106B was initially identified as a risk factor for FTLD, but recent studies highlighted its general role in neurodegenerative diseases. Very recently TMEM106B has also been characterized to regulate aging phenotypes. TMEM106B is a 274-residue lysosomal protein whose cytoplasmic domain functions in the endosomal/autophagy pathway by dynamically and transiently interacting with diverse categories of proteins but the underlying structural basis remains completely unknown. Here we conducted bioinformatics analysis and biophysical characterization by CD and NMR spectroscopy, and obtained results reveal that the TMEM106B cytoplasmic domain is intrinsically disordered with no well-defined three-dimensional structure. Nevertheless, detailed analysis of various multi-dimensional NMR spectra allowed defining residue-specific conformations and dynamics. Overall, the TMEM106B cytoplasmic domain is lacking of any tight tertiary packing and relatively flexible. However, several segments are populated with dynamic/nascent secondary structures and have relatively restricted backbone motions. In particular, the fragment Ser12-Met36 is highly populated with α- helix conformation. Our study thus decodes that being intrinsically disordered allows the TMEM106B cytoplasmic domain to dynamically and transiently interact with a variety of distinct partners.

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Conformation and membrane interaction studies of the potent antimicrobial and anticancer peptide palustrin-Ca

10.1101/2021.07.22.453375 ◽

2021 ◽

Author(s):

Patrick Brendan Timmons ◽

Chandralal M Hewage

Keyword(s):

Sodium Dodecyl ◽

Three Dimensional ◽

Dynamics Simulation ◽

Peptide Sequence ◽

Helical Conformation ◽

Dimensional Structure ◽

Amino Acid Residues ◽

Anticancer Activities ◽

American Bullfrog ◽

Α Helix

Palustrin-Ca (GFLDIIKDTGKEFAVKILNNLKCKLAGGCPP) is a host defense peptide with potent antimicrobial and anticancer activities, first isolated from the skin of the American bullfrog Lithobates catesbeianus. The peptide is 31 amino acid residues long, cationic and amphipathic. Two-dimensional NMR spectroscopy was employed to characterise its three-dimensional structure in a 50/50% water/2,2,2-trifluoroethanol-d3 mixture. The structure is defined by an α-helix that spans between Ile6-Ala26, and a cyclic disulphide bridged domain at the C-terminal end of the peptide sequence, between residues 23 and 29. A molecular dynamics simulation was employed to model the peptide's interactions with sodium dodecyl sulphate micelles, a widely used bacterial membrane-mimicking environment. Throughout the simulation, the peptide was found to maintain its α-helical conformation between residues Ile6-Ala26, while adopting a position parallel to the surface to micelle, which is energetically-favourable due to many hydrophobic and electrostatic contacts with the micelle.

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Evolutionary conservation of the intrinsic disorder-based Radical-Induced Cell Death1 hub interactome

Scientific Reports ◽

10.1038/s41598-019-55385-3 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Lise Friis Christensen ◽

Lasse Staby ◽

Katrine Bugge ◽

Charlotte O’Shea ◽

Birthe B. Kragelund ◽

...

Keyword(s):

Transcription Factor ◽

Stress Responses ◽

Plant Stress ◽

Three Dimensional ◽

Intrinsic Disorder ◽

Dimensional Structure ◽

Linear Motif ◽

Intrinsically Disordered ◽

Helix Formation ◽

Α Helix

AbstractRadical-Induced Cell Death1 (RCD1) functions as a cellular hub interacting with intrinsically disordered transcription factor regions, which lack a well-defined three-dimensional structure, to regulate plant stress. Here, we address the molecular evolution of the RCD1-interactome. Using bioinformatics, its history was traced back more than 480 million years to the emergence of land plants with the RCD1-binding short linear motif (SLiM) identified from mosses to flowering plants. SLiM variants were biophysically verified to be functional and to depend on the same RCD1 residues as the DREB2A transcription factor. Based on this, numerous additional members may be assigned to the RCD1-interactome. Conservation was further strengthened by similar intrinsic disorder profiles of the transcription factor homologs. The unique structural plasticity of the RCD1-interactome, with RCD1-binding induced α-helix formation in DREB2A, but not detectable in ANAC046 or ANAC013, is apparently conserved. Thermodynamic analysis also indicated conservation with interchangeability between Arabidopsis and soybean RCD1 and DREB2A, although with fine-tuned co-evolved binding interfaces. Interruption of conservation was observed, as moss DREB2 lacked the SLiM, likely reflecting differences in plant stress responses. This whole-interactome study uncovers principles of the evolution of SLiM:hub-interactions, such as conservation of α-helix propensities, which may be paradigmatic for disorder-based interactomes in eukaryotes.

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Structural Basis for Env Incorporation into HIV-1 Particles

Proceedings ◽

10.3390/proceedings2020050114 ◽

2020 ◽

Vol 50 (1) ◽

pp. 114

Author(s):

R. Elliot Murphy ◽

Alexandra B. Samal ◽

Gunnar Eastep ◽

Ruba H. Ghanam ◽

Peter E. Prevelige ◽

...

Keyword(s):

Structural Characterization ◽

Late Phase ◽

Three Dimensional ◽

Dimensional Structure ◽

Structural Basis ◽

Membrane Interactions ◽

Env Protein ◽

Gag Assembly ◽

Hiv 1

During the late phase of the HIV-1 replication cycle, the Gag polyproteins are transported to the plasma membrane (PM) for assembly. Gag targeting and assembly on the PM is dependent on interactions between its matrix (MA) domain and phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). Subsequent to Gag assembly, the envelope (Env) protein is recruited to the PM for incorporation into virus particles. Evidence suggests that the incorporation of the Env protein is mediated by interactions between the MA domain of Gag and the cytoplasmic tail of the gp41 subunit of Env (gp41CT), a mechanism that remains to be elucidated. Trimerization of the MA domain of Gag appears to be an obligatory step for this interaction. The interplay between gp41CT, the MA trimer, and the membrane has yet to be determined. Our lab has pioneered methods and approaches to investigate, at the molecular level, how the retroviral MA domains of Gag interact with membranes, a key requirement for understanding the Gag assembly and Env incorporation. Herein, we devised innovative approaches that will enable the structural characterization of the gp41CT–MA–membrane interactions. We employed structural biology (NMR and cryo-electron microscopy, biophysical methods, and biochemical tools to generate a macromolecular picture of how the MA domain of Gag binds to the membrane and how it interacts with gp41CT. To this end, we: (i) determined the three-dimensional structure of HIV-1 gp41CT and characterized its interaction with the membrane, (ii) engineered trimeric constructs of gp41CT and the MA to recapitulate the native and functional states of the proteins, and (iii) utilized membrane nanodisc technology to anchor the MA and gp41CT proteins. Our studies will allow for a detailed structural characterization of the gp41CT–MA–membrane interactions, which will advance our knowledge of HIV-1 Gag assembly and Env incorporation.

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Ribokinase fromLeishmania donovani: purification, characterization and X-ray crystallographic analysis

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x18000109 ◽

2018 ◽

Vol 74 (2) ◽

pp. 99-104 ◽

Cited By ~ 1

Author(s):

Santhosh Gatreddi ◽

Sayanna Are ◽

Insaf Ahmed Qureshi

Keyword(s):

Functional Characterization ◽

Three Dimensional ◽

Protozoan Parasite ◽

Crystallographic Analysis ◽

Size Exclusion ◽

Dimensional Structure ◽

Diffusion Method ◽

Gene Encoding ◽

Cell Parameters ◽

Α Helix

Leishmaniais an auxotrophic protozoan parasite which acquires D-ribose by transporting it from the host cell and also by the hydrolysis of nucleosides. The enzyme ribokinase (RK) catalyzes the first step of ribose metabolism by phosphorylating D-ribose using ATP to produce D-ribose-5-phosphate. To understand its structure and function, the gene encoding RK fromL. donovaniwas cloned, expressed and purified using affinity and size-exclusion chromatography. Circular-dichroism spectroscopy of the purified protein showed comparatively more α-helix in the secondary-structure content, and thermal unfolding revealed theTmto be 317.2 K. Kinetic parameters were obtained by functional characterization ofL. donovaniRK, and theKmvalues for ribose and ATP were found to be 296 ± 36 and 116 ± 9.0 µM, respectively. Crystals obtained by the hanging-drop vapour-diffusion method diffracted to 1.95 Å resolution and belonged to the hexagonal space groupP61, with unit-cell parametersa=b= 100.25,c= 126.77 Å. Analysis of the crystal content indicated the presence of two protomers in the asymmetric unit, with a Matthews coefficient (VM) of 2.45 Å3 Da−1and 49.8% solvent content. Further study revealed that human counterpart of this protein could be used as a template to determine the first three-dimensional structure of the RK from trypanosomatid parasites.

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Crystallization and preliminary crystallographic analysis of human muscle phosphofructokinase, the main regulator of glycolysis

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x14008723 ◽

2014 ◽

Vol 70 (5) ◽

pp. 578-582 ◽

Cited By ~ 4

Author(s):

Marco Kloos ◽

Antje Brüser ◽

Jürgen Kirchberger ◽

Torsten Schöneberg ◽

Norbert Sträter

Keyword(s):

Three Dimensional ◽

Crystallographic Analysis ◽

Human Muscle ◽

Yeast Cells ◽

Dimensional Structure ◽

Structural Basis ◽

Diffusion Method ◽

Rabbit Muscle ◽

Main Regulator ◽

Purification Protocol

Whereas the three-dimensional structure and the structural basis of the allosteric regulation of prokaryotic 6-phosphofructokinases (Pfks) have been studied in great detail, knowledge of the molecular basis of the allosteric behaviour of the far more complex mammalian Pfks is still very limited. The human muscle isozyme was expressed heterologously in yeast cells and purified using a five-step purification protocol. Protein crystals suitable for diffraction experiments were obtained by the vapour-diffusion method. The crystals belonged to space groupP6222 and diffracted to 6.0 Å resolution. The 3.2 Å resolution structure of rabbit muscle Pfk (rmPfk) was placed into the asymmetric unit and optimized by rigid-body and groupB-factor refinement. Interestingly, the tetrameric enzyme dissociated into a dimer, similar to the situation observed in the structure of rmPfk.

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Solution structure of the strawberry allergen Fra a 1

Bioscience Reports ◽

10.1042/bsr20120058 ◽

2012 ◽

Vol 32 (6) ◽

pp. 567-575 ◽

Cited By ~ 14

Author(s):

Christian Seutter von Loetzen ◽

Kristian Schweimer ◽

Wilfried Schwab ◽

Paul Rösch ◽

Olivia Hartl-Spiegelhauer

Keyword(s):

Solution Structure ◽

Three Dimensional ◽

Protein Family ◽

Dimensional Structure ◽

Hydrophobic Pocket ◽

Pigment Synthesis ◽

Family Protein ◽

Pr10 Protein ◽

Α Helix

The PR10 family protein Fra a 1E from strawberry (Fragaria x ananassa) is down-regulated in white strawberry mutants, and transient RNAi (RNA interference)-mediated silencing experiments confirmed that Fra a 1 is involved in fruit pigment synthesis. In the present study, we determined the solution structure of Fra a 1E. The protein fold is identical with that of other members of the PR10 protein family and consists of a seven-stranded antiparallel β-sheet, two short V-shaped α-helices and a long C-terminal α-helix that encompass a hydrophobic pocket. Whereas Fra a 1E contains the glycine-rich loop that is highly conserved throughout the protein family, the volume of the hydrophobic pocket and the size of its entrance are much larger than expected. The three-dimensional structure may shed some light on its physiological function and may help to further understand the role of PR10 proteins in plants.

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Structural insights into the cause of human RSPH4A primary ciliary dyskinesia

Molecular Biology of the Cell ◽

10.1091/mbc.e20-12-0806 ◽

2021 ◽

pp. mbc.E20-12-0806

Author(s):

Yanhe Zhao ◽

Justine Pinskey ◽

Jianfeng Lin ◽

Weining Yin ◽

Patrick R. Sears ◽

...

Keyword(s):

Primary Ciliary Dyskinesia ◽

Electron Tomography ◽

Three Dimensional ◽

Dimensional Structure ◽

Structural Basis ◽

Radial Spoke ◽

Radial Spokes ◽

Cilia And Flagella ◽

Tract Infections ◽

Ciliary Dyskinesia

Cilia and flagella are evolutionarily conserved eukaryotic organelles involved in cell motility and signaling. In humans, mutations in Radial Spoke Head Protein 4 homolog A ( RSPH4A) can lead to primary ciliary dyskinesia (PCD), a life-shortening disease characterized by chronic respiratory tract infections, abnormal organ positioning, and infertility. Despite its importance for human health, the location of RSPH4A in human cilia has not been resolved, and the structural basis of RSPH4A-/- PCD remains elusive. Here, we present the native, three-dimensional structure of RSPH4A-/- human respiratory cilia using samples collected non-invasively from a PCD patient. Using cryo-electron tomography and subtomogram averaging, we compared the structures of control and RSPH4A-/- cilia, revealing primary defects in two of the three radial spokes (RSs) within the axonemal repeat and secondary (heterogeneous) defects in the central pair complex. Similar to RSPH1-/- cilia, the radial spoke heads of RS1 and RS2, but not RS3, were missing in RSPH4A-/- cilia. However, RSPH4A-/- cilia also exhibited defects within the arch domains adjacent to the RS1 and RS2 heads, which were not observed with RSPH1 loss. Our results provide insight into the underlying structural basis for RSPH4A-/- PCD and highlight the benefits of applying cryo-ET directly to patient samples for molecular structure determination. [Media: see text]

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The Structure of Bypass of Forespore C, an Intercompartmental Signaling Factor during Sporulation in Bacillus

Journal of Biological Chemistry ◽

10.1074/jbc.m506910200 ◽

2005 ◽

Vol 280 (43) ◽

pp. 36214-36220 ◽

Cited By ~ 1

Author(s):

Hayley M. Patterson ◽

James A. Brannigan ◽

Simon M. Cutting ◽

Keith S. Wilson ◽

Anthony J. Wilkinson ◽

...

Keyword(s):

Rna Polymerase ◽

Mother Cell ◽

Asymmetric Cell Division ◽

Three Dimensional ◽

Dimensional Structure ◽

Coat Proteins ◽

Terminal Domain ◽

Spore Coat Proteins ◽

Α Helix ◽

Β Sheet

Sporulation in Bacillus subtilis begins with an asymmetric cell division giving rise to smaller forespore and larger mother cell compartments. Different programs of gene expression are subsequently directed by compartment-specific RNA polymerase σ-factors. In the final stages, spore coat proteins are synthesized in the mother cell under the control of RNA polymerase containing σK, (EσK). σK is synthesized as an inactive zymogen, pro-σK, which is activated by proteolytic cleavage. Processing of pro-σK is performed by SpoIVFB, a metalloprotease that resides in a complex with SpoIVFA and bypass of forespore (Bof)A in the outer forespore membrane. Ensuring coordination of events taking place in the two compartments, pro-σK processing in the mother cell is delayed until appropriate signals are received from the forespore. Cell-cell signaling is mediated by SpoIVB and BofC, which are expressed in the forespore and secreted to the intercompartmental space where they regulate pro-σK processing by mechanisms that are not yet fully understood. Here we present the three-dimensional structure of BofC determined by solution state NMR. BofC is a monomer made up of two domains. The N-terminal domain, containing a four-stranded β-sheet onto one face of which an α-helix is packed, closely resembles the third immunoglobulin-binding domain of protein G from Streptococcus. The C-terminal domain contains a three-stranded β-sheet and three α-helices in a novel domain topology. The sequence connecting the domains contains a conserved DISP motif to which mutations that affect BofC activity map. Possible roles for BofC in the σK checkpoint are discussed in the light of sequence and structure comparisons.

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Structural Basis of the Enhanced Pollutant-Degrading Capabilities of an Engineered Biphenyl Dioxygenase

Journal of Bacteriology ◽

10.1128/jb.00952-15 ◽

2016 ◽

Vol 198 (10) ◽

pp. 1499-1512 ◽

Cited By ~ 3

Author(s):

Sonali Dhindwal ◽

Leticia Gomez-Gil ◽

David B. Neau ◽

Thi Thanh My Pham ◽

Michel Sylvestre ◽

...

Keyword(s):

Polychlorinated Biphenyl ◽

Three Dimensional ◽

Binding Pocket ◽

Degradation Pathway ◽

Dimensional Structure ◽

Structural Basis ◽

Biphenyl Dioxygenase ◽

Pcb Congeners ◽

Biphenyl Degradation ◽

Insight Into

ABSTRACTBiphenyl dioxygenase, the first enzyme of the biphenyl catabolic pathway, is a major determinant of which polychlorinated biphenyl (PCB) congeners are metabolized by a given bacterial strain. Ongoing efforts aim to engineer BphAE, the oxygenase component of the enzyme, to efficiently transform a wider range of congeners. BphAEII9, a variant of BphAELB400in which a seven-residue segment,335TFNNIRI341, has been replaced by the corresponding segment of BphAEB356,333GINTIRT339, transforms a broader range of PCB congeners than does either BphAELB400or BphAEB356, including 2,6-dichlorobiphenyl, 3,3′-dichlorobiphenyl, 4,4′-dichlorobiphenyl, and 2,3,4′-trichlorobiphenyl. To understand the structural basis of the enhanced activity of BphAEII9, we have determined the three-dimensional structure of this variant in substrate-free and biphenyl-bound forms. Structural comparison with BphAELB400reveals a flexible active-site mouth and a relaxed substrate binding pocket in BphAEII9that allow it to bind different congeners and which could be responsible for the enzyme's altered specificity. Biochemical experiments revealed that BphAEII9transformed 2,3,4′-trichlorobiphenyl and 2,2′,5,5′-tetrachlorobiphenyl more efficiently than did BphAELB400and BphAEB356. BphAEII9also transformed the insecticide dichlorodiphenyltrichloroethane (DDT) more efficiently than did either parental enzyme (apparentkcat/Kmof 2.2 ± 0.5 mM−1s−1, versus 0.9 ± 0.5 mM−1s−1for BphAEB356). Studies of docking of the enzymes with these three substrates provide insight into the structural basis of the different substrate selectivities and regiospecificities of the enzymes.IMPORTANCEBiphenyl dioxygenase is the first enzyme of the biphenyl degradation pathway that is involved in the degradation of polychlorinated biphenyls. Attempts have been made to identify the residues that influence the enzyme activity for the range of substrates among various species. In this study, we have done a structural study of one variant of this enzyme that was produced by family shuffling of genes from two different species. Comparison of the structure of this variant with those of the parent enzymes provided an important insight into the molecular basis for the broader substrate preference of this enzyme. The structural and functional details gained in this study can be utilized to further engineer desired enzymatic activity, producing more potent enzymes.

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