The X-ray Structure of NccX from Cupriavidus metallidurans 31A Illustrates Potential Dangers of Detergent Solubilization When Generating and Interpreting Crystal Structures of Membrane Proteins

Ziani, Widade; Maillard, Antoine P.; Petit-Härtlein, Isabelle; Garnier, Norbert; Crouzy, Serge; Girard, Éric; Covès, Jacques

doi:10.1074/jbc.m114.586537

Cited by 10 publications

(6 citation statements)

References 58 publications

(53 reference statements)

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“…B. Model of the transmembrane domain of L. monocytogenes PgdA (residues 3–25) obtained with SWISS MODEL (Biasini et al ., ) on the nickel‐cobalt‐cadmium resistance protein NccX from Cupriavidus metallidurans as template (Ziani et al ., ). Residues contributing to PBP A1 binding (red) and PgdA dimerization (blue) are shown.…”

Section: Resultsmentioning

confidence: 98%

Stimulation of PgdA‐dependent peptidoglycan N‐deacetylation by GpsB‐PBP A1 in Listeria monocytogenes

Rismondo

Wamp

Aldridge

et al. 2017

Molecular Microbiology

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Listeria monocytogenes and other pathogenic bacteria modify their peptidoglycan to protect it against enzymatic attack through the host innate immune system, such as the cell wall hydrolase lysozyme. During our studies on GpsB, a late cell division protein that controls activity of the bi-functional penicillin binding protein PBP A1, we discovered that GpsB influences lysozyme resistance of L. monocytogenes as mutant strains lacking gpsB showed an increased lysozyme resistance. Deletion of pbpA1 corrected this effect, demonstrating that PBP A1 is also involved in this. Susceptibility to lysozyme mainly depends on two peptidoglycan modifying enzymes: The peptidoglycan N-deacetylase PgdA and the peptidoglycan O-acetyltransferase OatA. Genetic and biochemical experiments consistently demonstrated that the increased lysozyme resistance of the ΔgpsB mutant was PgdA-dependent and OatA-independent. Protein-protein interaction studies supported the idea that GpsB, PBP A1 and PgdA form a complex in L. monocytogenes and identified the regions in PBP A1 and PgdA required for complex formation. These results establish a physiological connection between GpsB, PBP A1 and the peptidoglycan modifying enzyme PgdA. To our knowledge, this is the first reported link between a GpsB-like cell division protein and factors important for escape from the host immune system.

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Section: Resultsmentioning

confidence: 98%

Stimulation of PgdA‐dependent peptidoglycan N‐deacetylation by GpsB‐PBP A1 in Listeria monocytogenes

Rismondo

Wamp

Aldridge

et al. 2017

Molecular Microbiology

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“…Moreover, recent work on the CnrX-homologue NccX has shown that these coalesced hydrophobic cores determine the dimerization of the full length, membrane-embedded protein as well. 46 Central in the CnrX protomer architecture is M123 that lies at the bottom of the cavity that harbors the metal ion. This residue is also crucial for the signaling mechanism as its recruitment in the coordination sphere of Ni(II) or Co(II) is the key event that initiates the biological response.…”

Section: Discussionmentioning

confidence: 99%

“…Events downstream metal sensing by CnrX remains to be investigated. Recent in vivo and in silico data have shown that the transmembrane segments of isolated NccX, a close CnrX homologue, engage in highly dynamic self-interactions, 46 which indicates that they might not support signaling on their own. The characterization of the interaction between CnrH and CnrY cytosolic domain also suggested that CnrY would probably not propagate a conformational change.…”

Section: Discussionmentioning

confidence: 99%

Response of CnrX from Cupriavidus metallidurans CH34 to nickel binding

et al. 2015

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Resistance to high concentration of nickel ions is mediated in Cupriavidus metallidurans by the CnrCBA transenvelope efflux complex. Expression of the cnrCBA genes is regulated by the transmembrane signal transduction complex CnrYXH. Together, the metal sensor CnrX and the transmembrane antisigma factor CnrY control the availability of the extracytoplasmic function sigma factor CnrH. Release of CnrH from sequestration by CnrY at the cytoplasmic side of the membrane depends essentially on the binding of the agonist metal ion Ni(ii) to the periplasmic metal sensor domain of CnrX. CnrH availability leads to transcription initiation at the promoters cnrYp and cnrCp and to the expression of the genes in the cnrYXHCBA nickel resistance determinant. The first steps of signal propagation by CnrX rely on subtle metal-dependent allosteric modifications. To study the nickel-mediated triggering process by CnrX, we have altered selected residues, F66, M123, and Y135, and explored the physiological consequences of these changes with respect to metal resistance, expression of a cnrCBA-lacZ reporter fusion and protein production. M123C- and Y135F-CnrXs have been further characterized in vitro by metal affinity measurements and crystallographic structure analysis. Atomic-resolution structures of metal-bound M123C- and Y135F-CnrXs showed that Ni(ii) binds two of the three canonical conformations identified and that Ni(ii) sensing likely proceeds by conformation selection.

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“…Another interesting case is the protein NccX, 359 of which a crystal structure has been determined in DPC. Intriguingly, “the periplasmic domains of NccX appeared collapsed against the hydrophobic TM segments, leading to an aberrant topology incompatible with membrane insertion.” The authors explained this unusual topology with a “detergent-induced redistribution of the hydrophobic interactions among the TM helices and a pair of hydrophobic patches keeping the periplasmic domains together in the native dimer.” In this case, alkyl phosphocholine led to a very unusual architecture that does not seem to be of biological relevance.…”

Section: Studies Of Mps In Dpc Reveal Strengths and Weaknessesmentioning

confidence: 99%

“…Monomeric single-span TM helices may not be impacted by these considerations, and in alkyl phosphocholine they may largely retain their structural properties (see the discussion on simulations of TM peptides in section 5 and references therein). This being said, the cases of NccX 359 and Rv1761c 358 show that also single-span helices may be significantly impacted in alkyl phosphocholine in terms of dimerization or local structure; the presence of hydrophilic or traditional helix breaking residues such as proline and glycine has led to an unphysiological structure in the latter case. Thus, even in single-span TM proteins, one needs to be cautious when interpreting structural data.…”

Section: Studies Of Mps In Dpc Reveal Strengths and Weaknessesmentioning

confidence: 99%

Perturbations of Native Membrane Protein Structure in Alkyl Phosphocholine Detergents: A Critical Assessment of NMR and Biophysical Studies

et al. 2018

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Membrane proteins perform a host of vital cellular functions. Deciphering the molecular mechanisms whereby they fulfill these functions requires detailed biophysical and structural investigations. Detergents have proven pivotal to extract the protein from its native surroundings. Yet, they provide a milieu that departs significantly from that of the biological membrane, to the extent that the structure, the dynamics, and the interactions of membrane proteins in detergents may considerably vary, as compared to the native environment. Understanding the impact of detergents on membrane proteins is, therefore, crucial to assess the biological relevance of results obtained in detergents. Here, we review the strengths and weaknesses of alkyl phosphocholines (or foscholines), the most widely used detergent in solution-NMR studies of membrane proteins. While this class of detergents is often successful for membrane protein solubilization, a growing list of examples points to destabilizing and denaturing properties, in particular for α-helical membrane proteins. Our comprehensive analysis stresses the importance of stringent controls when working with this class of detergents and when analyzing the structure and dynamics of membrane proteins in alkyl phosphocholine detergents.

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The X-ray Structure of NccX from Cupriavidus metallidurans 31A Illustrates Potential Dangers of Detergent Solubilization When Generating and Interpreting Crystal Structures of Membrane Proteins

Cited by 10 publications

References 58 publications

Stimulation of PgdA‐dependent peptidoglycan N‐deacetylation by GpsB‐PBP A1 in Listeria monocytogenes

Stimulation of PgdA‐dependent peptidoglycan N‐deacetylation by GpsB‐PBP A1 in Listeria monocytogenes

Response of CnrX from Cupriavidus metallidurans CH34 to nickel binding

Perturbations of Native Membrane Protein Structure in Alkyl Phosphocholine Detergents: A Critical Assessment of NMR and Biophysical Studies

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