Joseph Parello

Structure-based analysis of GPCR function: evidence for a novel pentameric assembly between the dimeric leukotriene B4 receptor BLT1 and the G-protein.

We produced human leukotriene B(4) (LTB(4)) receptor BLT1 as a recombinant protein in Escherichia coli. This detergent-solubilized receptor displays two states with regard to its affinity for LTB(4): (i) a low-affinity state (K(a)=7.8x10(8)M(-1)) that involves a receptor homodimer (BLT1.LTB(4))(2); we report evidence for a central role of the sixth transmembrane helix in regulating the stability of this homodimer; (ii) a high-affinity state (K(a)=1.3x10(10)M(-1)) upon interaction of the receptor with the heterotrimeric GDP-loaded G-protein, Galpha(i2)beta(1)gamma(2). Association of the G-protein with recombinant BLT1 induces GDP-GTP exchange by the Galpha subunit. These results indicate that isolated BLT1 is fully representative of the in vivo receptor with regard to high-affinity recognition of LTB(4), association with a G-protein and activation of Galpha. Using a combination of mass spectrometry after chemical cross-linking and neutron-scattering in solution with the native complex, we establish unambiguously that only one G-protein trimer binds to a receptor dimer to form the stoichiometrically defined (BLT1.LTB(4))(2):Galpha(i2)beta(1)gamma(2) pentameric assembly. This suggests that receptor dimerization could be crucial to transduction of the LTB(4)-induced signal.

Structure-based analysis of GPCR function: conformational adaptation of both agonist and receptor upon leukotriene B4 binding to recombinant BLT1.

We produced the human leukotriene B(4) (LTB(4)) receptor BLT1, a G-protein-coupled receptor, in Escherichia coli with yields that are sufficient for the first structural characterization of this receptor in solution. Overexpression was achieved through codon optimization and the search for optimal refolding conditions of BLT1 recovered from inclusion bodies. The detergent-solubilized receptor displays a 3D-fold compatible with a seven transmembrane (TM) domain with ca 50% alpha-helix and an essential disulfide bridge (circular dichroism evidence); it binds LTB(4) with K(a)=7.8(+/-0.2)x10(8)M(-1) and a stoichiometric ratio of 0.98(+/-0.02). Antagonistic effects were investigated using a synthetic molecule that shares common structural features with LTB(4). We report evidence that both partners, LTB(4) and BLT1, undergo a rearrangement of their respective conformations upon complex formation: (i) a departure from planarity of the LTB(4) conjugated triene moiety; (ii) a change in the environment of Trp234 (TM-VI helix) and in the exposure of the cytoplasmic region of this transmembrane helix.

Hydration-Coupled Dynamics in Proteins Studied by Neutron Scattering and NMR: The Case of the Typical EF-Hand Calcium-Binding Parvalbumin

The influence of hydration on the internal dynamics of a typical EF-hand calciprotein, parvalbumin, was investigated by incoherent quasi-elastic neutron scattering (IQNS) and solid-state 13C-NMR spectroscopy using the powdered protein at different hydration levels. Both approaches establish an increase in protein dynamics upon progressive hydration above a threshold that only corresponds to partial coverage of the protein surface by the water molecules. Selective motions are apparent by NMR in the 10-ns time scale at the level of the polar lysyl side chains (externally located), as well as of more internally located side chains (from Ala and Ile), whereas IQNS monitors diffusive motions of hydrogen atoms in the protein at time scales up to 20 ps. Hydration-induced dynamics at the level of the abundant lysyl residues mainly involve the ammonium extremity of the side chain, as shown by NMR. The combined results suggest that peripheral water-protein interactions influence the protein dynamics in a global manner. There is a progressive induction of mobility at increasing hydration from the periphery toward the protein interior. This study gives a microscopic view of the structural and dynamic events following the hydration of a globular protein.

Non-equivalence of the CD and EF sites of muscular parvalbumins. A 113Cd NMR study

The tertiary structure of parvalbumins is well documented by the determination of atomic coordinates for the component ~14.25 from carp muscle by X-ray crystallography [l] . A significant feature of this structure is the presence of two calcium ions at sites named CD and EF respectively [2] . Six oxygen atoms are liganded to the central cation in an octahedral arrangement, with Ca . . .O distances ranging from 2.15-2.85 A [l] . The first site contains solely protein ligands: four carboxyl groups, a peptide bond carbonyl group and a serine hydroxyl group. The site EF also contains four carboxyl groups and a carbonyl group but the sixth ligand is provided by a water molecule [l] , This suggests that the parvalbumin sites CD and EF will have different physicochemical and functional properties. Studies of the binding of Ca(I1) by various techniques have shown that parvalbumins have two cationic sites with high affinity for calcium ions [3-51, but have not been able to distinguish between the two sites. It has also been shown that many other cations compete for the ionic sites of parvalbumins [6-91. Specific binding of Na(1) [lo] and Mg(II) [ 1 l] has been observed using “Na and “Mg NMR spectroscopy. Kinetic aspects of the binding of Ca(II) to parvalbumins have been investigated directly by 43Ca NMR [ 121. Although well adapted to charac-

Conformational studies on muscular parvalbumins. II. Nuclear magnetic resonance analysis.

Summary NMR spectroscopy (proton resonance at 100 and 270 MHz) has been used to observe conformational features in muscular parvalbumins from hake (Merluccius merluccius) and carp (Cyprinus carpio), in the native state, in the denatured state (6M guanidinium chloride or heating) and after almost complete removal of calcium ions. From these observations it appears that the removal of the strongly bound calcium ions leads to a structure very similar to that obtained by chemical or thermal denaturation. On the other hand, the NMR spectrum of the native carp parvalbumins can be interpreted in the light of the recent X-Ray data obtained elsewhere for this protein. Some aspects of the primary structure of these parvalbumins, such as the presence of an N-terminal acetyl residue, have also been investigated with the NMR technique.

Mg2+ binding to parvalbumins studied by 25Mg and 113Cd NMR spectroscopy.

Parvalbumins (Pa) are low molecular weight (“11 500) proteins found in the muscles of most vertebrates. They have a strong affinity for Ca” [ 1,2]. It has been established by X-ray crystallographic studies of parvalbumin component ~14.25 from carp muscle that 2 Ca*+ occupy 2 sites within the protein molecule. These sites are called CD and EF [3] and they certainly correspond to the high-affinity Ca2+ sites (Kd -1 0e7 M) which were observed in solution [4]. The CD and EF sites are non-equivalent as shown by ‘r3Cd NMR spectroscopy studies of a Cd2’-loaded carp parvalbumin : two ‘13Cd signals with different chemical shifts are observed in the NMR spectrum [5]. Conformational studies of parvalbumins in solution have revealed that the tertiary structure of these globular proteins is dependent on the Ca2+ content [6-81. ‘H NMR spectroscopy clearly demonstrated that the highly compact structure of the 2 Ca*+ form is lost after removal of Ca*+ [6]. The Ca*+-free form corresponds to a less compact structure which is converted back to the initial one on addition of the missing Ca2+ [9]. Two different forms of the same parvalbumin molecule have been suggested to occur during the activation-relaxation cycle of the muscle [lo] . These forms might be related to the occurrence of different parvalbumin conformations depending on the Ca2+ concentration in the sarcoplasm. The interaction of Mg*’ might also play a role in the conformational transition of parvalbumins because of the relatively high concentration of Mg2+ in the muscle, i.e., 2-6 mM [ 1 I] which should be compared to 0.05-I mM protein [ 121.

Crystal structure determination and refinement of pike 4.10 parvalbumin (minor component from Esox lucius).

The crystal and molecular structure of the minor component of pike parvalbumins has been determined at 1.93 A resolution by molecular replacement (1 A = 0.1 nm). The crystals are orthorhombic, space group P2(1)2(1)2 with a = 59.62 A, b = 59.83 A and c = 26.35 A. A location of the secondary cation binding site is proposed for this parvalbumin of the beta phylogenetic series.

NMR studies of primary and secondary sites of parvalbumins using the two paramagnetic probes Gd (III) and Mn (II).

The binding of cations by parvalbumins was studied by the proton relaxation enhancement (PRE) method using the paramagnetic probes Gd(III) and Mn(II). Gd(III) appears as a specific probe of the primary sites CD and EF with the following binding parameters: n = 2, KdGd = 0.5 x 10(-11) M and epsilon b = 2.3. The low value of epsilon b is the result of a nearly complete dehydration of the protein bound ions. Competition experiments between Gd(III) and various diamagnetic cations show the following order of affinity for the EF and CD sites: Mg2+ less than Zn2+ less than Sr2+ less than Ca2+ less than Cd2+ less than La3+ less than or equal to Gd3+. Mn 2+ is a specific probe of a secondary site with the following binding parameters: n = 1, KdMn = 0.6 x 10(-3) M and epsilon b = 17. The high value of epsilon b suggests that the protein bound Mn(II) has retained most of its hydration shell. Competition experiments between (Mn(II) and different cations show similar affinities for this site: Ca2+ less than or equal to Mg2+ less than or equal to Cd2+ less than or equal to Mn2+. This secondary site is located near the EF primary site.

Spermidinyl-CoA-based HAT inhibitors block DNA repair and provide cancer-specific chemo- and radiosensitization

Acetyl group turnover on specific lysine ε-amino groups of the core chromosomal histones regulates DNA accessibility function, and the acetylating and deacetylating enzymes that govern the turnover provide important targets for the development of anti-cancer drugs. Histone deacetylase (HDAC) inhibitors have been developed and evaluated extensively in clinical trials, while the development of inhibitors of histone acetyltransferase (HAT) has proceeded more slowly. Here we have examined the cellular effects of an S-substituted coenzyme A (CoA) inhibitor of histone acetylation, consisting of spermidine (Spd) linked to the S-terminus of CoA through a thioglycolic acid linkage (adduct abbreviated as Spd-CoA), as well as the effects of a truncated Spd-CoA derivative lacking the negatively charged portion of the CoA moiety. While exposure of cancer cells to Spd-CoA has little effect on cell viability, it causes a rapid inhibition of histone acetylation that correlates with a transient arrest of DNA synthesis, a transient delay in S-phase progression, and an inhibition of nucleotide excision repair and DNA double strand break repair. These effects correlate with increased cellular sensitivity to the DNA-targeted chemotherapeutic drugs, cisplatin (Platinol™) and 5-Fluorouracil, to the DNA damaging drug, camptothecin, and to UV-C irradiation. The sensitization effects of Spd-CoA are not observed in normal cells due to a barrier to uptake. The truncated Spd-CoA derivative displays similar but enhanced chemosensitization effects, suggesting that further modifications of the Spd-CoA structure could further improve potency. The results demonstrate that Spd-CoA and its truncated version are efficiently and selectively internalized into cancer cells, and suggest that the resulting inhibition of acetylation-dependent DNA repair enhances cellular sensitivity to DNA damage. These and related inhibitors of histone acetylation could therefore constitute a novel class of potent therapy sensitizers applicable to a broad range of conventional cancer treatments.

The Cation-binding Domain from the α Subunit of Integrin α5β1 Is a Minimal Domain for Fibronectin Recognition

The cation-binding domain from the α subunit of human integrin α5β1 was produced as a recombinant protein, α5-(229–448). This protein displays a well defined fold with a content of 30–35% α-helix and 20–25% β-strand, based on circular dichroism. The binding of Ca2+ or Mg2+ to α5-(229–448) results in a biphasic conformational rearrangement consistent with the occurrence of two classes of cation-binding sites differing by their affinities. The two classes of sites are located in two conformationally independent lobes, as established by a parallel study of two recombinant half-domains (N- and C-terminal) that also adopt stable folds. Upon saturation with divalent cations, α5-(229–448) binds an Arg-Gly-Asp (RGD)-containing fibronectin ligand to form a 1:1 complex. Complex formation is associated with a specific conformational adaptation of the ligand, suggesting an induced fit mechanism. In contrast, neither of the half-domains is competent for ligand binding. The α5-(229–448)-fibronectin complex is dissociated in the presence of an RGD peptide, as well as of a simple carboxylic acid, suggesting that the RGD aspartyl carboxylate is an essential element that directly interacts with the α5 cation-binding domain.