The use of Co<sup>2+</sup>for crystallization and structure determination, using a conventional monochromatic X-ray source, of flax rust avirulence protein

Gunčar, Gregor; Wang, Ching-I. A.; Forwood, Jade K.; Teh, Trazel; Catanzariti, Ann‐Maree; Ellis, Jeffrey G.; Dodds, Peter N.; Kobe, Boštjan

doi:10.1107/s1744309107004599

Cited by 15 publications

(13 citation statements)

References 43 publications

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“…Multiple-scattering parameters for imidazole ligands bound to various metals were generated from crystallographic coordinates using the FEFF8 software package with previously published crystal structures as input (17, 31, 33–36). The best fits resulted in four prominent multiple-scattering features and paths of similar overall lengths were combined to make four imidazole scattering paths, matching these four prominent features as outlined by Costello et al (37, 38).…”

Section: Methodsmentioning

confidence: 99%

Effects of Select Histidine to Cysteine Mutations on Transcriptional Regulation by Escherichia coli RcnR

Higgins¹,

Hu²,

Chivers

et al. 2012

Biochemistry

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The RcnR metalloregulator represses the transcription of the Co(II) and Ni(II) exporter, RcnAB. Previous studies have shown that Co(II) and Ni(II) bind to RcnR in six-coordinate sites, resulting in de-repression. Here, the roles of His60, His64, and His67 in specific metal recognition are examined. His60 and His64 correspond to ligands that are important for Cu(I) binding in the homologous Cu(I)-responsive metalloregulator, CsoR. These residues are known to be functionally important in RcnR transcriptional regulation. XAS was used to examine the structure of bound cognate and non-cognate metal ions, and lacZ reporter assays were used to assess the transcription of rcnA in response to metal binding in the three His → Cys mutations, H60C, H64C and H67C. These studies confirm that both Ni(II) and Co(II) use His64 as a ligand. H64C-RcnR is also the only known mutation that retains a Co(II) response while eliminating the response to Ni(II) binding. XAS data indicate that His60 and His67 are potential Co(II) ligands. The effects of the mutations of His60, His64, and His67 residues on the structures of the non-cognate metal ions (Zn(II) and Cu(I)) reveals that these residues have distinctive roles in binding non-cognate metals. None of the His → Cys mutants in RcnR confer any response to Cu(I) binding, including H64C-RcnR, where the ligands involved in Cu(I) binding in CsoR are present. These data indicate that while the secondary, tertiary and quaternary structures of CsoR and RcnR are quite similar, small changes in primary sequence reveal that the specific mechanisms involved in metal recognition are quite different.

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Section: Methodsmentioning

confidence: 99%

Effects of Select Histidine to Cysteine Mutations on Transcriptional Regulation by Escherichia coli RcnR

Higgins¹,

Hu²,

Chivers

et al. 2012

Biochemistry

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“…Both proteins contained N-terminal hexahistidine and ubiquitin tags, enabling the proteins to be purified using Co 2þ affinity chromatography, and the tags were removed using the deubiquitinating enzyme . The structure of AvrL567-A was determined by single-wavelength anomalous dispersion techniques using a home x-ray source, taking advantage of a bound Co 2þ ion (Guncar et al, 2007). The structure of (A) Sequence alignment of the known members of the AvrL567 family.…”

Section: Structure Determinationmentioning

confidence: 99%

“…The crystals have the symmetry of the orthorhombic space group P2 1 2 1 2 1 with unit cell lengths of a ¼ 39.6 Å , b ¼ 52.2 Å , and c ¼ 70.9 Å and one molecule per asymmetric unit. Initially, a data set was collected at a maximum resolution of 2.0 Å and used to solve the structure by single anomalous dispersion phasing (Guncar et al, 2007); a data set collected at a maximum resolution of 1.4 Å was used for refinement. Briefly, the presence of Co 2þ was detected using the program Hyss (Grosse-Kunstleve and Adams, 2003), which showed one Co 2þ binding site with ;100% occupancy.…”

Section: Crystallization and Crystal Structure Determinationmentioning

confidence: 99%

Crystal Structures of Flax Rust Avirulence Proteins AvrL567-A and -D Reveal Details of the Structural Basis for Flax Disease Resistance Specificity

et al. 2007

Self Cite

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The gene-for-gene mechanism of plant disease resistance involves direct or indirect recognition of pathogen avirulence (Avr) proteins by plant resistance (R) proteins. Flax rust (Melampsora lini) AvrL567 avirulence proteins and the corresponding flax (Linum usitatissimum) L5, L6, and L7 resistance proteins interact directly. We determined the three-dimensional structures of two members of the AvrL567 family, AvrL567-A and AvrL567-D, at 1.4-and 2.3-Å resolution, respectively. The structures of both proteins are very similar and reveal a b-sandwich fold with no close known structural homologs. The polymorphic residues in the AvrL567 family map to the surface of the protein, and polymorphisms in residues associated with recognition differences for the R proteins lead to significant changes in surface chemical properties. Analysis of single amino acid substitutions in AvrL567 proteins confirm the role of individual residues in conferring differences in recognition and suggest that the specificity results from the cumulative effects of multiple amino acid contacts. The structures also provide insights into possible pathogen-associated functions of AvrL567 proteins, with nucleic acid binding activity demonstrated in vitro. Our studies provide some of the first structural information on avirulence proteins that bind directly to the corresponding resistance proteins, allowing an examination of the molecular basis of the interaction with the resistance proteins as a step toward designing new resistance specificities.

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“…1c). CBD proteolytic digests in the presence of cobalt, a known modulator [40–44], and in the presence of both cobalt and calcium were also analyzed using MALDI-TOF MS and were compared to apo- and calcium-bound CBD proteolysis results.…”

Section: Introductionmentioning

confidence: 99%

Probing the 3-D Structure, Dynamics, and Stability of Bacterial Collagenase Collagen Binding Domain (apo- versus holo-) by Limited Proteolysis MALDI-TOF MS

Sides

Liyanage

Lay

et al. 2011

J. Am. Soc. Mass Spectrom.

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Pairing limited proteolysis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to probe clostridial collagenase collagen binding domain (CBD) reveals the solution dynamics and stability of the protein, as these factors are crucial to CBD effectiveness as a drug-delivery vehicle. MS analysis of proteolytic digests indicates initial cleavage sites, thereby specifying the less stable and highly accessible regions of CBD. Modulation of protein structure and stability upon metal binding is shown through MS analysis of calcium-bound and cobalt-bound CBD proteolytic digests. Previously determined X-ray crystal structures illustrate that calcium binding induces secondary structure transformation in the highly mobile N-terminal arm and increases protein stability. MS-based detection of exposed residues confirms protein flexibility, accentuates N-terminal dynamics, and demonstrates increased global protein stability exported by calcium binding. Additionally, apo- and calcium-bound CBD proteolysis sites correlate well with crystallographic B-factors, accessibility, and enzyme specificity. MS-observed cleavage sites with no clear correlations are explained either by crystal contacts of the X-ray crystal structures or by observed differences between Molecules A and B in the X-ray crystal structures. The study newly reveals the absence of the βA strand and thus the very dynamic N-terminal linker, as corroborated by the solution X-ray scattering results. Cobalt binding has a regional effect on the solution phase stability of CBD, as limited proteolysis data implies the capture of an intermediate-CBD solution structure when cobalt is bound.

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The use of Co²⁺for crystallization and structure determination, using a conventional monochromatic X-ray source, of flax rust avirulence protein

Cited by 15 publications

References 43 publications

Effects of Select Histidine to Cysteine Mutations on Transcriptional Regulation by Escherichia coli RcnR

Effects of Select Histidine to Cysteine Mutations on Transcriptional Regulation by Escherichia coli RcnR

Crystal Structures of Flax Rust Avirulence Proteins AvrL567-A and -D Reveal Details of the Structural Basis for Flax Disease Resistance Specificity

Probing the 3-D Structure, Dynamics, and Stability of Bacterial Collagenase Collagen Binding Domain (apo- versus holo-) by Limited Proteolysis MALDI-TOF MS

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The use of Co2+for crystallization and structure determination, using a conventional monochromatic X-ray source, of flax rust avirulence protein

Cited by 15 publications

References 43 publications

Effects of Select Histidine to Cysteine Mutations on Transcriptional Regulation by Escherichia coli RcnR

Effects of Select Histidine to Cysteine Mutations on Transcriptional Regulation by Escherichia coli RcnR

Crystal Structures of Flax Rust Avirulence Proteins AvrL567-A and -D Reveal Details of the Structural Basis for Flax Disease Resistance Specificity

Probing the 3-D Structure, Dynamics, and Stability of Bacterial Collagenase Collagen Binding Domain (apo- versus holo-) by Limited Proteolysis MALDI-TOF MS

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The use of Co²⁺for crystallization and structure determination, using a conventional monochromatic X-ray source, of flax rust avirulence protein