Backbone structure of a small helical integral membrane protein: A unique structural characterization

Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306-4390, USA.
Protein Science (Impact Factor: 2.85). 01/2008; 18(1):134-46. DOI: 10.1002/pro.24
Source: PubMed


The structural characterization of small integral membrane proteins pose a significant challenge for structural biology because of the multitude of molecular interactions between the protein and its heterogeneous environment. Here, the three-dimensional backbone structure of Rv1761c from Mycobacterium tuberculosis has been characterized using solution NMR spectroscopy and dodecylphosphocholine (DPC) micelles as a membrane mimetic environment. This 127 residue single transmembrane helix protein has a significant (10 kDa) C-terminal extramembranous domain. Five hundred and ninety distance, backbone dihedral, and orientational restraints were employed resulting in a 1.16 A rmsd backbone structure with a transmembrane domain defined at 0.40 A. The structure determination approach utilized residual dipolar coupling orientation data from partially aligned samples, long-range paramagnetic relaxation enhancement derived distances, and dihedral restraints from chemical shift indices to determine the global fold. This structural model of Rv1761c displays some influences by the membrane mimetic illustrating that the structure of these membrane proteins is dictated by a combination of the amino acid sequence and the protein's environment. These results demonstrate both the efficacy of the structural approach and the necessity to consider the biophysical properties of membrane mimetics when interpreting structural data of integral membrane proteins and, in particular, small integral membrane proteins.

Download full-text


Available from: Richard C Page
  • Source
    • "All probes cause line-broadening in signals originating from proximal amide atoms located within the same domain. PRE-derived distance restraints were obtained as described in detail by Sattler and colleagues using an apparent molecular correlation time of 16 ns for the electron-nucleus vector [33] [34] [35]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Staphylococcus aureus is a medically important bacterial pathogen that during infections acquires iron from human hemoglobin (Hb). It uses two closely related iron regulated surface determinant (Isd) proteins to capture and extract the oxidized form of heme (hemin) from Hb, IsdH and IsdB. Both receptors rapidly extract hemin using a conserved tri-domain unit consisting of two NEAr iron Transporter (NEAT) domains connected by a helical linker domain. To gain insight into the mechanism of extraction we used NMR to investigate the structure and dynamics of the 38.8kDa tri-domain IsdH protein (IsdH(N2N3), A326-D660 with a Y642A mutation that prevents hemin binding). The structure was modeled using long-range paramagnetic relaxation enhancement (PRE) distance restraints, dihedral angle, small angle x-ray scattering, residual dipolar coupling and inter-domain NOE data. The receptor adopts an extended conformation wherein the linker and N3 domains pack against each other via a hydrophobic interface. In contrast, the N2 domain contacts the linker domain via a hydrophilic interface, and based on NMR relaxation data undergoes inter-domain motions enabling it to reorient with respect to the body of the protein. Ensemble calculations were used to estimate the range of N2 domain positions compatible with the PRE data. A comparison of the Hb-free and -bound forms reveals that Hb binding alters the positioning of the N2 domain. We propose that binding occurs through a combination of conformational selection and induced fit mechanisms that may promote hemin release from Hb by altering the position of its F-helix. Copyright © 2015. Published by Elsevier Ltd.
    Full-text · Article · Feb 2015 · Journal of Molecular Biology
    • "Rv1761c His, C-terminus TEV protease pMCSG7 2K3M [130] Integrin 3 TM Ketosteroid isomerase Asp-Pro site pET31b 2RN0 [37] Twin Arginine Trans- port His, C-terminus - pET21a 2L16 [131] "
    [Show abstract] [Hide abstract]
    ABSTRACT: Membrane proteins play important roles in signal transduction across the cell membrane. Structural information for the membrane proteins is still limited due to many technical challenges. Membrane proteins containing a single α- helical transmembrane (TM) domain are very important in several pathways. Solution NMR spectroscopy is an important tool for the study of the structure of the TM domain of these types of proteins due to their small size. In this review, we summarize the importance of some single-span membrane proteins in signal transduction and the importance of understanding the structure of the TM domain. We discussed the current progress in the structural study of these types of proteins using solution NMR spectroscopy. We summarize the structures solved during last several years. The structures of the regulatory domain of the ion channels such as KCNE1, integrin and viral proteins such as the M2 channel are described. The binding interface of single TM-TM domains is discussed based on NMR structural studies. Strategies including sample preparation, detergent screening, and structural determination of single-span membrane protein are summarized. We also discuss the potential application of NMR spectroscopy to drug discovery of proteins with a single-span TM domain.
    No preview · Article · Oct 2012 · Current Protein and Peptide Science
  • Source
    • "); FXYD1 (Teriete et al., 2007); KCNE1 (Kang et al., 2008); DsbB (Zhou et al., 2008); DAGK, diacylglycerol kinase (Van Horn et al., 2009); Rv1761c (Page et al., 2009); ArcB, QseC, KdpD (Maslennikov et al., 2010); Presenilin-1 C-terminal fragment (Sobhanifar et al., 2010); UCP2, uncoupling protein 2 (Berardi et al., 2011); Proteorhodopsin (Reckel et al., 2011). b Number of conformational restraints used in the structure calculation, as reported in the structural statistics table of the corresponding publication. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Nuclear magnetic resonance (NMR) structure calculations of the α-helical integral membrane proteins DsbB, GlpG, and halorhodopsin show that distance restraints from paramagnetic relaxation enhancement (PRE) can provide sufficient structural information to determine their structure with an accuracy of about 1.5 Å in the absence of other long-range conformational restraints. Our systematic study with simulated NMR data shows that about one spin label per transmembrane helix is necessary for obtaining enough PRE distance restraints to exclude wrong topologies, such as pseudo mirror images, if only limited other NMR restraints are available. Consequently, an experimentally realistic amount of PRE data enables α-helical membrane protein structure determinations that would not be feasible with the very limited amount of conventional NOESY data normally available for these systems. These findings are in line with our recent first de novo NMR structure determination of a heptahelical integral membrane protein, proteorhodopsin, that relied extensively on PRE data.
    Full-text · Article · May 2012 · Structure
Show more