Kinase-active Signaling Complexes of Bacterial Chemoreceptors Do Not Contain Proposed Receptor−Receptor Contacts Observed in Crystal Structures

Fowler, Daniel J.; Weis, Robert M.; Thompson, Lynmarie K.

doi:10.1021/bi901565k

Cited by 24 publications

(62 citation statements)

References 58 publications

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“…For the serine receptor Thompson and coworkers have measured an intra-dimer distance of 8.8Å between a unique Cys residue and a 5F-Phe residue. [••73] For models of the kinase-active signaling complex the predicted inter-dimer distances were much shorter, however the REDOR measurements indicated the same distance as that measured previously for the intra-dimer subunit of the complex suggesting that the high protein density of the crystal lattice may be influencing the structure of this functional state.…”

Section: Solid-state Nmr Of Protein-protein Interactionssupporting

confidence: 62%

Solid state NMR and protein–protein interactions in membranes

Miao

Cross

2013

Current Opinion in Structural Biology

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Solid state NMR spectroscopy has evolved rapidly in recent years into an excellent tool for the characterization of membrane proteins and their complexes. In the past few years it has also become clear that the structure of membrane proteins, especially helical membrane proteins is determined, in part, by the membrane environment. Therefore, the modeling of this environment by a liquid crystalline lipid bilayer for solid state NMR has generated a unique tool for the characterization of native conformational states, local and global dynamics, and high resolution structure for these proteins. Protein-protein interactions can also benefit from this solid state NMR capability to characterize membrane proteins in a native-like environment. These complexes take the form of oligomeric structures and hetero-protein interactions both with water soluble proteins and other membrane proteins.

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Section: Solid-state Nmr Of Protein-protein Interactionssupporting

confidence: 62%

Solid state NMR and protein–protein interactions in membranes

Miao

Cross

2013

Current Opinion in Structural Biology

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“…13 C S 0 and S 1 rotational-echo double-resonance (REDOR) spectra were acquired [22,23]. The reduction in 13 CO intensity in the S 1 spectrum relative to S 0 was used to probe proximity of labeled ( lab ) and natural abundance ( na ) 13 CO nuclei to either lab FP-HP 15 N or membrane headgroup 31 P nuclei.…”

Section: Methodsmentioning

confidence: 99%

pH-dependent vesicle fusion induced by the ectodomain of the human immunodeficiency virus membrane fusion protein gp41: Two kinetically distinct processes and fully-membrane-associated gp41 with predominant β sheet fusion peptide conformation

Ratnayake

Sackett

Nethercott

et al. 2015

Biochimica et Biophysica Acta (BBA) - Biomembranes

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The gp41 protein of the Human Immunodeficiency Virus (HIV) catalyzes fusion between HIV and host cell membranes. The ~180-residue ectodomain of gp41 is outside the virion and is the most important gp41 region for membrane fusion. The ectodomain consists of an apolar fusion peptide (FP) region followed by N-heptad repeat (NHR), loop, and C-heptad repeat (CHR) regions. The FP is critical for fusion and is hypothesized to bind to the host cell membrane. Large ectodomain constructs either with or without the FP are highly aggregated at physiologic pH but soluble in the pH 3–4 range with hyperthermostable hairpin structure with antiparallel NHR and CHR helices. The present study focuses on the large gp41 ectodomain constructs “Hairpin” (HP) containing NHR+loop+CHR and “FP-Hairpin” (FP-HP) containing FP+NHR+loop+CHR. Both proteins induce rapid and extensive fusion of anionic vesicles at pH 4 where the protein is positively-charged but do not induce fusion at pH 7 where the protein is negatively charged. This observation, along with lack of fusion of neutral vesicles at either pH supports the significance of attractive protein/membrane electrostatics in fusion. The functional role of the hydrophobic FP is supported by increases in the rate and extent of fusion for FP-HP relative to HP. There are two kinetically distinct fusion processes at pH 4: (1) a faster ~100 ms−1 process with rate strongly positively correlated with vesicle charge; and (2) a slower ~5 ms−1 process with extent strongly inversely correlated with this charge. The faster charge-dependent process is likely related to the electrostatic energy released upon initial monomer protein binding to the vesicle. After dissipation of this energy, the subsequent slower process is likely due to the equilibrium membrane-associated structure of the protein. The slower process may be more physiologically relevant because HIV/host cell fusion occurs at physiologic pH with gp41 restricted to the narrow region between the two membranes. Previous solid-state NMR (SSNMR) of membrane-associated FP-HP has supported protein oligomers with FP’s in an intermolecular antiparallel β sheet. There was an additional population of molecules with α helical FPs and the samples likely contained a mixture of membrane-bound and -unbound protein. For the present study, samples were prepared with fully membrane-bound FP-HP and subsequent SSNMR showed dominant β FP conformation at both low and neutral pH. The fusogenic structure is likely β for the slow and perhaps the fast process. SSNMR also showed close contact of the FP with the lipid headgroups at both low and neutral pH whereas the NHR+CHR regions had contact at low pH and were more distant at neutral pH, consistent with the protein/membrane electrostatics.

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“…40–43 Functional complexes of CF, CheA, and CheW bound to vesicles were prepared as previously described, 33 using excess CheA and CheW to drive incorporation of all of the CF into complexes. 42 These complexes were characterized using kinase and sedimentation assays. 31 Details of these protocols are described in the Supplementary Information.…”

Section: Methodsmentioning

confidence: 99%

Hydrogen Exchange Mass Spectrometry of Functional Membrane-Bound Chemotaxis Receptor Complexes

et al. 2013

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The transmembrane signaling mechanism of bacterial chemotaxis receptors is thought to involve changes in receptor conformation and dynamics. The receptors function in ternary complexes with two other proteins, CheA and CheW, that form extended membrane-bound arrays. Previous studies have shown that attractant binding induces a small (~2 Å) piston displacement of one helix of the periplasmic and transmembrane domains towards the cytoplasm, but it is not clear how this signal propagates through the cytoplasmic domain to control the kinase activity of the CheA bound at the membrane-distal tip, nearly 200 Å away. The cytoplasmic domain has been shown to be highly dynamic, which raises the question of how a small piston motion could propagate through a dynamic domain to control CheA kinase activity. To address this, we have developed a method for measuring dynamics of the receptor cytoplasmic fragment (CF) in functional complexes with CheA and CheW. Hydrogen exchange mass spectrometry (HDX-MS) measurements of global exchange of CF demonstrate that CF exhibits significantly slower exchange in functional complexes than in solution. Since the exchange rates in functional complexes are comparable to that of other proteins of similar structure, the CF appears to be a well-structured protein within these complexes, which is compatible with its role in propagating a signal that appears to be a tiny conformational change in the periplasmic and transmembrane domains of the receptor. We also demonstrate the feasibility of this protocol for local exchange measurements, by incorporating a pepsin digest step to produce peptides with 87% sequence coverage and only 20% back exchange. This method extends HDX-MS to membrane-bound functional complexes without detergents that may perturb the stability or structure of the system.

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Kinase-active Signaling Complexes of Bacterial Chemoreceptors Do Not Contain Proposed Receptor−Receptor Contacts Observed in Crystal Structures

Cited by 24 publications

References 58 publications

Solid state NMR and protein–protein interactions in membranes

Solid state NMR and protein–protein interactions in membranes

pH-dependent vesicle fusion induced by the ectodomain of the human immunodeficiency virus membrane fusion protein gp41: Two kinetically distinct processes and fully-membrane-associated gp41 with predominant β sheet fusion peptide conformation

Hydrogen Exchange Mass Spectrometry of Functional Membrane-Bound Chemotaxis Receptor Complexes

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