Structural characterisation of the HT3 motif of the polyhistidine triad protein D from <i>Streptococcus pneumoniae</i>

Luo, Zhenyao; Pederick, Victoria G.; Paton, James C.; McDevitt, Christopher A.; Kobe, Boštjan

doi:10.1002/1873-3468.13122

Cited by 5 publications

(6 citation statements)

References 44 publications

(74 reference statements)

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“…Although the Pht proteins have been refractory to high-resolution structure determination of the full-length protein, truncated domains containing individual HT motifs (HT1, HT2, and HT3) have been revealed (13,16,27). Structural analyses have suggested that each of the HT motifs of PhtD adopt a similar fold, with an equivalent capacity for Zn 2ϩ binding (13). Nonetheless, functional studies have shown that HT1 has the most critical role in zinc acquisition (15).…”

Section: Discussionmentioning

confidence: 99%

“…Here, the Pht proteins are shown to be cell wall-associated proteins that contribute to zinc acquisition via AdcAII, albeit with overlapping functionality (8,11,12). PhtD is the most highly conserved Pht family protein across pneumococcal strains and contains five HT motifs, with each motif predicted to bind zinc (13)(14)(15). Recent studies have shown that the HT1 motif, which is closest to the cell membrane, is the most important for the contribution of PhtD to zinc acquisition (15).…”

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confidence: 95%

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Defining the Role of the Streptococcus agalactiae Sht-Family Proteins in Zinc Acquisition and Complement Evasion

et al. 2019

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Streptococcus agalactiae is not only part of the human intestinal and urogenital microbiota but is also a leading cause of septicemia and meningitis in neonates. Its ability to cause disease depends upon the acquisition of nutrients from its environment, including the transition metal ion zinc. The primary zinc acquisition system of the pathogen is the Adc/Lmb ABC permease, which is essential for viability in zinc-restricted environments. Here, we show that in addition to the AdcCB transporter and the three zinc-binding proteins, Lmb, AdcA, and AdcAII, S. agalactiae zinc homeostasis also involves two streptococcal histidine triad (Sht) proteins. Sht and ShtII are required for zinc uptake via the Lmb and AdcAII proteins with apparent overlapping functionality and specificity. Both Sht-family proteins possess fivehistidine triad motifs with similar hierarchies of importance for Zn homeostasis. Independent of its contribution to zinc homeostasis, Sht has previously been reported to bind factor H leading to predictions of a contribution to complement evasion. Here, we investigated ShtII to ascertain whether it had similar properties. Analysis of recombinant Sht and ShtII reveals that both proteins have similar affinities for factor H binding. However, neither protein aided in resistance to complement in human blood. These findings challenge prior inferences regarding the in vivo role of the Sht proteins in resisting complement᎑mediated clearance. IMPORTANCE This study examined the role of the two streptococcal histidine triad (Sht) proteins of Streptococcus agalactiae in zinc homeostasis and complement resistance. We showed that Sht and ShtII facilitate zinc homeostasis in conjunction with the metal-binding proteins Lmb and AdcAII. Here, we show that the Sht-family proteins are functionally redundant with overlapping roles in zinc uptake. Further, this work reveals that although the Sht-family proteins bind to factor H in vitro this did not influence survival in human blood.

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Section: Discussionmentioning

confidence: 99%

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confidence: 95%

Defining the Role of the Streptococcus agalactiae Sht-Family Proteins in Zinc Acquisition and Complement Evasion

et al. 2019

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“…terminal domain of polyhistidine triad protein D (PhtD-C) protein [13] and a P450 enzyme [unpublished].…”

Section: A C C E P T E D Accepted Manuscriptmentioning

confidence: 99%

A new vector coupling ligation-independent cloning with sortase a fusion for efficient cloning and one-step purification of tag-free recombinant proteins

Jia

Crawford

Zhang

et al. 2019

Protein Expression and Purification

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We have developed a new ligation independent cloning (LIC) vector -pSrtA9, which can be utilized for one-step purification of recombinant proteins. The new LIC site in the pSrtA9 vector, hosts a DNA sequence centered on a SfoI restriction site and integrates a coding sequence for sortase A (SrtA) recognition. Preceding the LIC site, pSrtA9 incorporates an N-terminal 6xHistag and the catalytic core of SrtA from Staphylococcus aureus (SrtA∆59). Thus, after cloning Highlights • pSrtA9 is a new ligation independent cloning (LIC) vector incorporating sortase A (SrtA) fusion and recognition site.• A fusion protein expressed from pSrtA9 features an N-terminal SrtA domain with a four amino acids linker for increased cleavage efficiency.• pSrtA9 allows efficient cloning and rapid purification of tag-free protein in a single step.

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“…As an electron has a charge, there is a magnetic moment associated with the spin angular momentum: where e  is the Bohr magneton, Ŝ is the spin angular momentum, and the factor g can be obtained in accordance with Dirac's relativistic quantum mechanics. The free electron g-value is ge = 2.00231930436153 (53).…”

Section: Electron Spin and Spin Hamiltonianmentioning

confidence: 99%

“…Interestingly, AdcA-mediated Zn 2+ homeostasis does not show a significant dependency on the polyhistidine triad (Pht) proteins (from 90 to 116 kDa in size) [53], in contrast with AdcAII-mediated Zn 2+ homeostasis [54] where the proteins help to facilitate Zn 2+ acquisition. Pht proteins exist on the cell surface and are implicated in the Zn 2+ transport pathway but their role is poorly understood [53].…”

Section: Role Of Adca Zn 2+ -Binding Protein For S Pneumoniae Zn 2+mentioning

confidence: 99%

Applications of electron paramagnetic resonance in biomedicine

Motygullina¹

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Spectroscopy and imaging are widely used to characterise systems structurally and functionally, and this information allows for the rational development of novel drugs and theranostics in many diverse areas of medicine. This thesis focuses on applying Electron Paramagnetic Resonance (EPR) techniques to determine the structure and function of protein molecules. A feasibility study to develop paramagnetic probes for EPR imaging is also presented.In EPR the probe is a paramagnetic centre and information is obtained by measuring interactions of this probe with its environment. In this thesis continuous wave (CW) EPR and Double Electron Electron Resonance (DEER) in conjunction with a site-directed spin labelling (SDSL) were employed to study structure and function of spin-labelled protein molecules. The techniques allow examination of molecular systems that cannot be crystallised or are too big for efficient NMR investigation. DEER provides information on distance distributions between two spin labels located on Cys-mutated residues of the protein molecule and can access longer distances in comparison with NMR. CW EPR provides information about the mobility of a single spin label which can be used to characterise protein dynamics. Structural modelling was used to combine the crystal structures and Molecular Dynamics (MD) simulations with DEER distance constraints to present a DEER-based structural model of the conformational variability of the protein molecule. Chapters 2 and 3 focus on characterising two metal ion substrate binding proteins (SBP), Zn 2+binding AdcA and Mn 2+ -binding PsaA, using DEER and CW EPR. The ATP-Binding Cassette (ABC) permeases, with which the SBPs are associated, are a primary importer used by bacteria to scavenge the essential first-row metal ions (e.g. iron, zinc, manganese) from a host environment. The process is essential for bacterial survival and propagation. Understanding the metal ion acquisition mechanisms by these SBPs can provide new opportunities for targeted drug development.Collectively, the CW EPR and DEER data along with crystal structures, differential scanning fluorimetry (DSF), Molecular Dynamics simulation, and smFRET microscopy, were able to determine a structural model for Zn 2+ -binding in the AdcA protein referred to as "trap-door" mechanism. Our data show that the "trap-door" mechanism employed by AdcA is different from the "spring-hammer" mechanism employed by PsaA.Non-Ribosomal Peptide Synthetase (NRPS) are the topic of chapter 4. NRPS's are a family of mega enzymes that produce a diverse range of pharmaceuticals, yet how these molecular machines operate to produce such complex chemicals is very poorly understood. This project will investigate module 7 of the teicoplanin NRPS, a 200 kDa protein, which is the last step in the production of the glycopeptide antibiotics (GPA). A comprehensive structural and functional understanding of the teicoplanin NRPS machinery will provide a paradigm for this enzyme family, enabling the tailored iv Publications included in this t...

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Structural characterisation of the HT3 motif of the polyhistidine triad protein D from Streptococcus pneumoniae

Cited by 5 publications

References 44 publications

Defining the Role of the Streptococcus agalactiae Sht-Family Proteins in Zinc Acquisition and Complement Evasion

Defining the Role of the Streptococcus agalactiae Sht-Family Proteins in Zinc Acquisition and Complement Evasion

A new vector coupling ligation-independent cloning with sortase a fusion for efficient cloning and one-step purification of tag-free recombinant proteins

Applications of electron paramagnetic resonance in biomedicine

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