Najmoutin G. Abdulaev

Phototransduction: crystal clear

Vertebrate visual phototransduction represents one of the best-characterized G-protein-coupled receptor-mediated signaling pathways. Structural analyses of rhodopsin, G protein, arrestin and several other phototransduction components have revealed common folds and motifs that are important for function. Static and dynamic information has been acquired through the application of X-ray diffraction, solution and solid-state nuclear magnetic resonance spectroscopys, electron and atomic force microscopys, and a host of indirect structural methods. A comprehensive understanding of phototransduction requires further structural work on individual components and their relevant complexes in solution and the native disk membrane. Given the accelerated pace of structure determination, it is anticipated that this will be the first G-protein-coupled pathway for which a complete molecular description is ultimately available.

Conformational Changes Associated with Receptor-stimulated Guanine Nucleotide Exchange in a Heterotrimeric G-protein α-Subunit NMR ANALYSIS OF GTPγS-BOUND STATES

Solution NMR studies of a 15N-labeled G-protein α-subunit (Gα) chimera (15N-ChiT)-reconstituted heterotrimer have shown previously that G-protein βγ-subunit (Gβγ) association induces a “pre-activated” conformation that likely facilitates interaction with the agonist-activated form of a G-protein-coupled receptor (R*) and guanine nucleotide exchange (Abdulaev, N. G., Ngo, T., Zhang, C., Dinh, A., Brabazon, D. M., Ridge, K. D., and Marino, J. P. (2005) J. Biol. Chem. 280, 38071-38080). Here we demonstrated that the 15N-ChiT-reconstituted heterotrimer can form functional complexes under NMR experimental conditions with light-activated, detergent-solubilized rhodopsin (R*), as well as a soluble mimic of R*. NMR methods were used to track R*-triggered guanine nucleotide exchange and release of guanosine 5′-O-3-thiotriphosphate (GTPγS)/Mg2+-bound ChiT. A heteronuclear single quantum correlation (HSQC) spectrum of R*-generated GTPγS/Mg2+-bound ChiT revealed 1HN, 15N chemical shift changes relative to GDP/Mg2+-bound ChiT that were similar, but not identical, to those observed for the \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{GDP}{\cdot}\mathrm{AlF}_{4}^{-}{/}\mathrm{Mg}^{2+}\) \end{document}-bound state. Line widths observed for R*-generated GTPγS/Mg2+-bound 15N-ChiT, however, indicated that it is more conformationally dynamic relative to the GDP/Mg2+- and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{GDP}{\cdot}\mathrm{AlF}_{4}^{-}{/}\mathrm{Mg}^{2+}\) \end{document}-bound states. The increased dynamics appeared to be correlated with Gβγ and R* interactions because they are not observed for GTPγS/Mg2+-bound ChiT generated independently of R*. In contrast to R*, a soluble mimic that does not catalytically interact with G-protein (Abdulaev, N. G., Ngo, T., Chen, R., Lu, Z., and Ridge, K. D. (2000) J. Biol. Chem. 275, 39354-39363) is found to form a stable complex with the GTPγS/Mg2+-exchanged heterotrimer. The HSQC spectrum of 15N-ChiT in this complex displays a unique chemical shift pattern that nonetheless shares similarities with the heterotrimer and GTPγS/Mg2+-bound ChiT. Overall, these results demonstrated that R*-induced changes in Gα can be followed by NMR and that guanine nucleotide exchange can be uncoupled from heterotrimer dissociation.

Functionally Discrete Mimics of Light-activated Rhodopsin Identified through Expression of Soluble Cytoplasmic Domains

Numerous studies on the seven-helix receptor rhodopsin have implicated the cytoplasmic loops and carboxyl-terminal region in the binding and activation of proteins involved in visual transduction and desensitization. In our continuing studies on rhodopsin folding, assembly, and structure, we have attempted to reconstruct the interacting surface(s) for these proteins by inserting fragments corresponding to the cytoplasmic loops and/or the carboxyl-terminal tail of bovine opsin either singly, or in combination, onto a surface loop in thioredoxin. The purpose of the thioredoxin fusion is to provide a soluble scaffold for the cytoplasmic fragments thereby allowing them sufficient conformational freedom to fold to a structure that mimics the protein-binding sites on light-activated rhodopsin. All of the fusion proteins are expressed to relatively high levels in Escherichia coli and can be purified using a two- or three-step chromatography procedure. Biochemical studies show that some of the fusion proteins effectively mimic the activated conformation(s) of rhodopsin in stimulating G-protein or competing with the light-activated rhodopsin/G-protein interaction, in supporting phosphorylation of the carboxyl-terminal opsin fragment by rhodopsin kinase, and/or phosphopeptide-stimulated arrestin binding. These results suggest that specific segments of the cytoplasmic surface of rhodopsin can adopt functionally discrete conformations in the absence of the connecting transmembrane helices and retinal chromophore.

Heterotrimeric G-protein α-Subunit Adopts a “Preactivated” Conformation When Associated with βγ-Subunits

Activation of a heterotrimeric G-protein by an agonist-stimulated G-protein-coupled receptor requires the propagation of structural signals from the receptor binding interface to the guanine nucleotide binding pocket of the G-protein. To probe the molecular basis of this signaling process, we are applying high resolution NMR to track structural changes in an isotope-labeled, full-length G-protein α-subunit (Gα) chimera (ChiT) associated with G-protein βγ-subunit (Gβγ) and activated receptor (R*) interactions. Here, we show that ChiT can be functionally reconstituted with Gβγ as assessed by aluminum fluoride-dependent changes in intrinsic tryptophan fluorescence and light-activated rhodopsin-catalyzed guanine nucleotide exchange. We further show that 15N-ChiT can be titrated with Gβγ to form stable heterotrimers at NMR concentrations. To assess structural changes in ChiT upon heterotrimer formation, HSQC spectra of the 15N-ChiT-reconstituted heterotrimer have been acquired and compared with spectra obtained for GDP/Mg2+-bound 15N-ChiT in the presence and absence of aluminum fluoride and guanosine 5′-3-O-(thio)triphosphate (GTPγS)/Mg2+-bound 15N-ChiT. As anticipated, Gβγ association with 15N-ChiT results in 1HN, 15N chemical shift changes relative to the GDP/Mg2+-bound state. Strikingly, however, most 1HN, 15N chemical shift changes associated with heterotrimer formation are the same as those observed upon formation of the \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{GDP}{\cdot}\mathrm{Al}\mathrm{F}_{4}^{-}{/}\mathrm{Mg}^{2+}\) \end{document}- and GTPγS/Mg2+-bound states. Based on these comparative analyses, assembly of the heterotrimer appears to induce structural changes in the switch II and carboxyl-terminal regions of Gα (“preactivation”) that may facilitate the interaction with R* and subsequent GDP/GTP exchange.

NMR analysis of rhodopsin–transducin interactions

Heterotrimeric G-protein activation by an agonist-stimulated G-protein coupled receptor (R*) requires the propagation of structural signals from the receptor interacting surfaces to the guanine nucleotide-binding pocket. Employing high-resolution NMR methods, we are probing heterotrimer-associated and rhodopsin-stimulated changes in an isotope-labeled G-protein alpha-subunit (G(alpha)). A key aspect of the work involves the trapping and interrogation of discrete R*-bound conformations of G(alpha). Our results demonstrate that functionally important changes in G(alpha) structure and dynamics can be detected and characterized by NMR, enabling the generation of robust models for the global and local structural changes accompanying signal transfer from R* to the G-protein.

Grafting Segments from the Extracellular Surface of CCR5 onto a Bacteriorhodopsin Transmembrane Scaffold Confers HIV-1 Coreceptor Activity

Components from the extracellular surface of CCR5 interact with certain macrophage-tropic strains of human immunodeficiency virus type 1 (HIV-1) to mediate viral fusion and entry. To mimic these viral interacting site(s), the amino-terminal and extracellular loop segments of CCR5 were linked in tandem to form concatenated polypeptides, or grafted onto a seven-transmembrane bacteriorhodopsin scaffold to generate several chimeras. The chimera studies identified specific regions in CCR5 that confer HIV-1 coreceptor function, structural rearrangements in the transmembrane region that may modulate this activity, and a role for the extracellular surface in folding and assembly. Methods developed here may be applicable to the dissection of functional domains from other seven-transmembrane receptors and form a basis for future structural studies.

Building a stage for interhelical play in rhodopsin

Biochemical data providing new insights into the packing of helices I and II in the transmembrane domain of rhodopsin reveals the existence of a specific set of size- and charge-sensitive interhelical interactions that influence protein tertiary structure. These findings have broad implications towards understanding the molecular consequences of naturally occurring mutations associated with the retinal degenerative disease autosomal dominant retinitis pigmentosa.

Folding and assembly in rhodopsin. Effect of mutations in the sixth transmembrane helix on the conformation of the third cytoplasmic loop.

Previous studies on bovine opsin folding and assembly have identified an amino-terminal fragment, EF(1–232), which folds and inserts into a membrane only after coexpression with its complementary carboxyl-terminal fragment, EF(233–348). To further characterize this interaction, EF(1–232) production was examined upon coexpression with carboxyl-terminal fragments of varying length and/or amino acid composition. These included fragments with incremental deletions of the third cytoplasmic loop (TH(241–348) and EF(249–348)), a fragment composed of the third cytoplasmic loop and sixth transmembrane helix (HF(233–280)), a fragment composed of the sixth and seventh transmembrane helices (FG(249–312)), and EF(233–348) and TH(241–348) fragments with Pro-267 or Trp-265 mutations. Although EF(1–232) production was independent of the third cytoplasmic loop and carboxyl-terminal tail, both the sixth and seventh transmembrane helices were essential. The effects of mutations in the sixth transmembrane helix on EF(1–232) expression were dependent on the length of the third cytoplasmic loop. Although Pro-267 mutations in EF(233–348) failed to stabilize EF(1–232) expression, their introduction into TH(241–348) was without discernible effects. However, Trp-265 substitutions in the EF(233–348) and TH(241–348) fragments conferred significant EF(1–232) production. Therefore, key residues in the transmembrane helices may exert their effects on opsin folding, assembly, and/or function by influencing the conformation of the connecting loops.

The three-dimensional structures of two isoforms of nucleoside diphosphate kinase from bovine retina.

The crystal structures of two isoforms of nucleoside diphosphate kinase from bovine retina overexpressed in Escherischia coli have been determined to 2.4 A resolution. Both the isoforms, NBR-A and NBR-B, are hexameric and the fold of the monomer is in agreement with NDP-kinase structures from other biological sources. Although the polypeptide chains of the two isoforms differ by only two residues, they crystallize in different space groups. NBR-A crystallizes in space group P212121 with an entire hexamer in the asymmetric unit, while NBR-B crystallizes in space group P43212 with a trimer in the asymmetric unit. The highly conserved nucleotide-binding site observed in other nucleoside diphosphate kinase structures is also observed here. Both NBR-A and NBR-B were crystallized in the presence of cGMP. The nucleotide is bound with the base in the anti conformation. The NBR-A active site contained both cGMP and GDP each bound at half occupancy. Presumably, NBR-A had retained GDP (or GTP) from the purification process. The NBR-B active site contained only cGMP.

Designing Point Mutants to Detect Structural Coupling in a Heterotrimeric G Protein α‐subunit by NMR Spectroscopy†

To better understand the mechanism by which the activating signal is transmitted from the receptor‐interacting regions on the G protein α‐subunit (Gα) to the guanine nucleotide‐binding pocket, we generated and characterized mutant forms of Gα with alterations in switch II (Trp‐207→Phe) and the carboxyl‐terminus (Phe‐350→Ala). Previously reported bacterial expression methods for the high‐level production of a uniformly isotope‐labeled Gtα/Gi1α chimera, ChiT, were successfully used to isolate milligram quantities of 15N‐labeled mutant protein. NMR analysis showed that while the GDP/Mg2+‐bound state of both mutants shared an overall conformation similar to that of the GDP/Mg2+‐bound state of ChiT, formation of the “transition/activated” state in the presence of aluminum fluoride (AlF4−) revealed distinct differences between the wild‐type and mutant Gα subunits, particularly in the response of the 1HN, 15N cross‐peak for the Trp‐254 indole in the Trp‐207→Phe mutant and the 1HN, 15N cross‐peak for Ala‐350 in the Phe‐350→Ala mutant. Consistent with the NMR data, the F350→Ala mutant showed an increase in intrinsic fluorescence that was similar to Gtα and ChiT upon formation of the “transition/activated” state in the presence of AlF4−, whereas the intrinsic fluorescence of the Trp‐207→Phe mutant decreased. These results show that the substitution of key amino acid positions in Gα can effect structural changes that may compromise receptor interactions and GDP/GTP exchange.