Complex Salt Bridges in Proteins: Statistical Analysis of Structure and Function

Musafia, Boaz; Buchner, Virginia; Arad, Dorit

doi:10.1006/jmbi.1995.0653

Cited by 174 publications

(158 citation statements)

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“…55-D6.30 Salt Bridge Calculations-Musafia et al (48) have reported that the average interatomic N-O distance for a simple Arg-Asp salt bridge is 2.93 Å. All measurements of salt bridge distances were made using Visual Molecular Dynamics (49).…”

Section: Methodsmentioning

confidence: 99%

A Lipid Pathway for Ligand Binding Is Necessary for a Cannabinoid G Protein-coupled Receptor

Hurst

Grossfield

Lynch

et al. 2010

Journal of Biological Chemistry

197

242

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The cannabinoid receptors belong to the class A (rhodopsin family) of G protein-coupled receptors (GPCRs).2 The second cannabinoid receptor subtype, CB2 (2), is highly expressed throughout the immune system (3, 4) and has been described in the central nervous system under both pathological (5) and physiological conditions (6). All known CB2 ligands are highly lipophilic. In fact, the CB2 endogenous cannabinoid, sn-2-arachidonoylglycerol (2-AG) (7,8), is synthesized on demand from the lipid bilayer itself in a two-step process in which phospholipase C-␤ hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol, which is then hydrolyzed by diacylglycerol lipase to yield 2-AG (9, 10). After 2-AG interaction with the membraneembedded CB receptor, it is hydrolyzed to arachidonic acid and glycerol by a membrane-associated enzyme, monoacylglycerol lipase (11). As revealed by the crystal structures of rhodopsin (12-15), the ␤ 2 -adrenergic receptor (AR) (16 -18), ␤ 1 -AR (19), and adenosine A2A receptor (20), the general topology of a GPCR includes the following: 1) an extracellular (EC) N terminus; 2) seven transmembrane ␣-helices (TMHs) arranged to form a closed bundle; 3) loops connecting TMHs that extend intra-and extracellularly; and 4) an intracellular (IC) C terminus that begins with a short helical segment (helix 8) oriented parallel to the membrane surface. Agonists bind inside the crevice formed by the TMH bundle and produce conformational changes on the IC face of the receptor that uncover previously masked G protein-binding sites (21), which then lead to G protein coupling. Biophysical studies using a variety of techniques indicate that ligand-induced receptor activation produces the following changes: 1) a conformational change in the W6.48 "toggle switch" within the ligand binding pocket (22); 2) a change in the relative orientations of TMH3 and -6 that breaks an IC "ionic lock" (23-28), with the intracellular end of TMH6 moving away from TMH3 by hinging and moving up toward lipid (27); 3) the uptake of two protons (29); and 4) an influx of water (30).Recent isothiocyanate covalent labeling studies have suggested that a classical cannabinoid, AM841, enters the CB2 receptor via the lipid bilayer (1). However, the sequence of steps involved in such a lipid pathway entry has not yet been elucidated. We report here microsecond time scale unbiased

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Section: Methodsmentioning

confidence: 99%

A Lipid Pathway for Ligand Binding Is Necessary for a Cannabinoid G Protein-coupled Receptor

Hurst

Grossfield

Lynch

et al. 2010

Journal of Biological Chemistry

197

242

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“…Although the role of simple salt bridges in providing protein stabilization is controversial (43,44), the importance of complex salt bridges in providing better stabilization than the sum of two individual salt bridges and the importance of arginine in complex salt bridges have been recognized (37,45).…”

Section: A Connection Between the 50-kda Upper And N-terminal Subdomamentioning

confidence: 99%

Myosin subfragment 1 structures reveal a partially bound nucleotide and a complex salt bridge that helps couple nucleotide and actin binding

Risal

Gourinath

Himmel

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Structural studies of myosin have indicated some of the conformational changes that occur in this protein during the contractile cycle, and we have now observed a conformational change in a bound nucleotide as well. The 3.1-Å x-ray structure of the scallop myosin head domain (subfragment 1) in the ADP-bound near-rigor state (lever arm Ϸ45°to the helical actin axis) shows the diphosphate moiety positioned on the surface of the nucleotide-binding pocket, rather than deep within it as had been observed previously. This conformation strongly suggests a specific mode of entry and exit of the nucleotide from the nucleotide-binding pocket through the so-called ''front door.'' In addition, using a variety of scallop structures, including a relatively high-resolution 2.75-Å nucleotide-free near-rigor structure, we have identified a conserved complex salt bridge connecting the 50-kDa upper and N-terminal subdomains. This salt bridge is present only in crystal structures of muscle myosin isoforms that exhibit a strong reciprocal relationship (also known as coupling) between actin and nucleotide affinity.M yosin is a motor protein that transduces ATP hydrolysis into mechanical work, leading to its translocation along filamentous actin. It is believed that the small conformational changes induced by enzymatic activity in the myosin ''motor domain'' (MD) are amplified by the motion of the ''lever arm.'' Three weak actin-binding (actin-detached) states have been identified crystallographically for scallop myosin subfragment 1 (S1) (1). These and other structures reveal that the motor function of myosin S1 is coordinated by three subdomains (50-kDa upper and lower subdomains and the converter) that rotate as rigid bodies around the relatively stable N-terminal subdomain (1-3). These rotations depend on conformational changes in three flexible joints (switch II, relay, and the SH1 helix) between the subdomains. Three-dimensional reconstructions of electron microscopic images of actin decorated by S1 of several myosin isoforms (4-6) have led to the suggestion that actin binds to portions of the 50-kDa upper and lower subdomains, whereas various crystal structures (see, for example, refs. 3 and 7-9) indicate that ATP binds at the opposite side of the myosin head in a pocket between the 50-kDa upper and Nterminal subdomains (Figs. 1 and 2A).One of the three structural conformations, the so-called ''prepower stroke,'' corresponds biochemically to the ADP⅐P i transition state (1,8,(10)(11)(12). Here, S1 displays a primed lever arm (Ϸ90°to the actin filament axis), closure of the 50-kDa cleft's base (which is near the nucleotide-binding pocket), and a bent switch II that interacts with both the nucleotide and switch I (a loop in the 50-kDa upper subdomain that coordinates the nucleotide). After this state, the tight binding of myosin to actin results in the so-called ''power stroke'' (after which the lever arm is Ϸ45°to the actin filament), a process believed to be initiated by the near-complete closing of the 50-kDa cleft (4-6). This...

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“…The simplest salt bridge that can be introduced would be a pair of side chains such as ER spaced at an interval (i, i + 4). E is preferred to D because of its more favorable helix propensity, while R is preferred to K because it has a larger positive surface that favors complex salt bridging (Musafia et al, 1995). A K + R substitution pattern has been identified in thermophilic proteins (O'Fagain, 1995).…”

Section: Abstract: Gcn4; Leucine Zipper; Salt Bridge; Thermal Stabilitymentioning

confidence: 99%

Surface salt bridges stabilize the GCN4 leucine zipper

Spek

Bui

et al. 1998

Protein Science

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We present a study of the role of salt bridges in stabilizing a simplified tertiary structural motif, the coiled-coil. Changes in GCN4 sequence have been engineered that introduce trial patterns of single and multiple salt bridges at solvent exposed sites. At the same sites, a set of alanine mutants was generated to provide a reference for thermodynamic analysis of the salt bridges. Introduction of three alanines stabilizes the dimer by 1.1 kcal/mol relative to the wild-type. An arrangement corresponding to a complex type of salt bridge involving three groups stabilizes the dimer by 1.7 kcall mol, an apparent elevation of the melting temperature relative to wild type of about 22 "C. While identifying local from nonlocal contributions to protein stability is difficult, stabilizing interactions can be identified by use of cycles. Introduction of alanines for side chains of lower helix propensity and complex salt bridges both stabilize the coiled-coil, so that combining the two should yield melting temperatures substantially higher than the starting species, approaching those of thermophilic sequences. Keywords: GCN4; leucine zipper; salt bridge; thermal stabilityInteractions that stabilize the native state of proteins include the hydrophobic effect, van der Waals interactions, hydrogen bonds and ionic effects, including dipole interactions and salt bridges (Creighton, 1993). The question of which of these are most important in protein stabilization has been debated since the review by Kauzmann (1959). One aspect of the problem concerns how to account for the additional stabilization of proteins from thermophiles, which can have very high thermal stabilities (see Hiller et al., 1997). Since the pioneering work of Matthews et al. (1974) on thermolysin, structures of thermophilic and mesophilic proteins have been compared in a search for clues to what accounts for the higher stability of the former (Korndorfer et al., 1995;Yip et al., 1995;Hatanaka et al., 1997;Robb & Maeder, 1998). In 1978, Perutz (1978 observed that the main discernible difference between a thermophilic and mesophilic version of ferrodoxin lay in the greater number of salt bridges on the surface of the thermophile. As more crystal structures of thermophilic proteins have become available, other mechanisms have been proposed to explain their stability (Vogt & Argos, 1997): improved internal packing, burial of a greater hydrophobic area (Chan et al., 1995; Delboni et al., 1995), and networks of complex salt bridges (Yip et al., 1995;Pappenberger et al., 1997 Yip et al., 1995;Robb & Maeder, 1998).Here, we consider the role of complex salt bridges in stabilizing a simplified model protein structure. The strength of a salt bridge can be estimated by different experimental methodologies: changes in the helicity of model peptides (Merutka & Stellwagen, 1990;Lyu et al., 1992;Scholtz et al., 1993), shifts in pK, of interacting side chains (Anderson et al., 1990;, or T, differences in model proteins Dao-Pin et al., 1991). Using the first method, Smi...

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Complex Salt Bridges in Proteins: Statistical Analysis of Structure and Function

Cited by 174 publications

References 0 publications

A Lipid Pathway for Ligand Binding Is Necessary for a Cannabinoid G Protein-coupled Receptor

A Lipid Pathway for Ligand Binding Is Necessary for a Cannabinoid G Protein-coupled Receptor

Myosin subfragment 1 structures reveal a partially bound nucleotide and a complex salt bridge that helps couple nucleotide and actin binding

Surface salt bridges stabilize the GCN4 leucine zipper

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