Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

18 pags., 11 figs.Particulate Guanylyl Cyclase Receptor A (pGC-A) is a natriuretic peptide membrane receptor, playing a vital role in controlling cardiovascular, renal, and endocrine functions. The extracellular domain interacts with natriuretic peptides and triggers the intracellular guanylyl cyclase domain to convert GTP to cGMP. To effectively develop methods to regulate pGC-A, structural information on the full-length form is needed. However, structural data on the transmembrane and intracellular domains are lacking. This work presents expression and optimization using baculovirus, along with the first purification of functional full-length human pGC-A. In vitro assays revealed the pGC-A tetramer was functional in detergent micelle solution. Based on our purification results and previous findings that dimer formation is required for functionality, we propose a tetramer complex model with two functional subunits. Previous research suggested pGC-A signal transduction is an ATP-dependent, two-step mechanism. Our results show the binding ligand also moderately activates pGC-A, and ATP is not crucial for activation of guanylyl cyclase. Furthermore, crystallization of full-length pGC-A was achieved, toward determination of its structure. Needle-shaped crystals with 3 Å diffraction were observed by serial crystallography. This work paves the road for determination of the full-length pGC-A structure and provides new information on the signal transduction mechanism.Tis project was supported by an award to J.C.B. and P.F. from the Mayo/ASU Structural Biology Alliance and by the Biodesign Center for Applied Structural Discovery at Arizona State University. Tis research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Ofce of Science user facility operated for the DOE Ofce of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357.Peer reviewe

Purification and Biophysical Characterization of the CapA Membrane Protein FTT0807 from <i>Francisella tularensis</i>

The <i>capA</i> gene (FTT0807) from Francisella tularensis subsp. tularensis SCHU S4 encodes a 44.4 kDa integral membrane protein composed of 403 amino acid residues that is part of an apparent operon that encodes at least two other membrane proteins, CapB, and CapC, which together play a critical role in the virulence and pathogenesis of this bacterium. The <i>capA</i> gene was overexpressed in Escherichia coli as a C-terminal His₆-tagged fusion with a folding reporter green fluorescent protein (frGFP). Purification procedures using several detergents were developed for the fluorescing and membrane-bound product, yielding approximately 30 mg of pure protein per liter of bacterial culture. Dynamic light scattering indicated that CapA-frGFP was highly monodisperse, with a size that was dependent upon both the concentration and choice of detergent. Circular dichroism showed that CapA-frGFP was stable over the range of 3–9 for the pH, with approximately half of the protein having well-defined α-helical and β-sheet secondary structure. The addition of either sodium chloride or calcium chloride at concentrations producing ionic strengths above 0.1 M resulted in a small increase of the α-helical content and a corresponding decrease in the random-coil content. Secondary-structure predictions on the basis of the analysis of the sequence indicate that the CapA membrane protein has two transmembrane helices with a substantial hydrophilic domain. The hydrophilic domain is predicted to contain a long disordered region of 50–60 residues, suggesting that the increase of α-helical content at high ionic strength could arise because of electrostatic interactions involving the disordered region. CapA is shown to be an inner-membrane protein and is predicted to play a key cellular role in the assembly of polysaccharides

Biophysical Characterization of a Vaccine Candidate against HIV-1: The Transmembrane and Membrane Proximal Domains of HIV-1 gp41 as a Maltose Binding Protein Fusion

<div><p>The membrane proximal region (MPR, residues 649–683) and transmembrane domain (TMD, residues 684–705) of the gp41 subunit of HIV-1’s envelope protein are highly conserved and are important in viral mucosal transmission, virus attachment and membrane fusion with target cells. Several structures of the trimeric membrane proximal external region (residues 662–683) of MPR have been reported at the atomic level; however, the atomic structure of the TMD still remains unknown. To elucidate the structure of both MPR and TMD, we expressed the region spanning both domains, MPR-TM (residues 649–705), in <i>Escherichia coli</i> as a fusion protein with maltose binding protein (MBP). MPR-TM was initially fused to the C-terminus of MBP via a 42 aa-long linker containing a TEV protease recognition site (MBP-linker-MPR-TM). Biophysical characterization indicated that the purified MBP-linker-MPR-TM protein was a monodisperse and stable candidate for crystallization. However, crystals of the MBP-linker-MPR-TM protein could not be obtained in extensive crystallization screens. It is possible that the 42 residue-long linker between MBP and MPR-TM was interfering with crystal formation. To test this hypothesis, the 42 residue-long linker was replaced with three alanine residues. The fusion protein, MBP-AAA-MPR-TM, was similarly purified and characterized. Significantly, both the MBP-linker-MPR-TM and MBP-AAA-MPR-TM proteins strongly interacted with broadly neutralizing monoclonal antibodies 2F5 and 4E10. With epitopes accessible to the broadly neutralizing antibodies, these MBP/MPR-TM recombinant proteins may be in immunologically relevant conformations that mimic a pre-hairpin intermediate of gp41.</p></div

Binding rate constants of MBP-linker-MPR-TM derived from SPR analysis^a.

<p>^aResults are the average of four independent measurements and are listed as mean ± SD.</p><p>Binding rate constants of MBP-linker-MPR-TM derived from SPR analysis<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136507#t001fn001" target="_blank">^a</a>.</p

Surface plasmon resonance analysis of MBP-linker-MPR-TM.

<p>The mAbs 2F5 and 4E10 were immobilized onto the surface of a gold chip (Plexera) and the purified MBP-linker-MPR-TM protein was the analyte. MBP (fractions A2 from SEC purification, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136507#pone.0136507.g001" target="_blank">Fig 1C</a>) was used as a negative control.</p

DLS measurement of 2 mg/ml MBP-linker-MPR-TM indicated monodispersity.

<p>DLS measurement of 2 mg/ml MBP-linker-MPR-TM indicated monodispersity.</p

CD, DLS and SPR measurements of MBP-AAA-MPR-TM.

<p>(A) Comparison of CD spectra of MBP-linker-MPR-TM and MBP-AAA-MPR-TM. (B) DLS measurement of 1 mg/ml MBP-AAA-MPR-TM showed one monodisperse peak at 7.4 ± 0.8 nm. (C) SPR analysis. The mAbs 2F5 and 4E10 were immobilized onto the surface of a gold chip (Plexera) and the purified MBP-AAA-MPR-TM protein was the analyte.</p

MALDI-TOF MS analysis of elutions from SEC.

<p>(A) MALDI-TOF MS analysis of the peak A1 from SEC (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136507#pone.0136507.g001" target="_blank">Fig 1C</a>). (B) MALDI-TOF MS analysis of the peak A2 from SEC (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136507#pone.0136507.g001" target="_blank">Fig 1C</a>). (C) Schematic representation of the MBP-linker-MPR-TM protein and predicted molecular weights of protein fragments. Residues 414–420 in green: TEV protease recognition site.</p