Structure of G-protein (RGS4) and methods of identifying agonists and antagonists using same

Abstract

The present invention provides a solution structure of free RGS4 determined using NMR techniques. The structure includes a G alpha binding site and allosteric bindingsite. The structural information provided can be employed to identify, select or design agonists and antagonists of RGS4 activity. The invention includes two dimensional and three dimensional models and representations of the structure of free RGS4 based upon structural coordinates that are provided that are useful in the methods described.

Description

Background of the invention

[0001] A wide variety of biochemical processes, especially those involving protein-protein interactions, are thought to be mediated by induced conformational changes in protein targets. The resulting structural changes in a protein can then be used to explain changes in its function (eg enzymatic activity) or its affinity for another protein in a biological system. Conformational changes have been proposed to occur in eukaryotic cells in cascades associated with steps in certain signal transduction pathways. Ubiquitous components of such signal transduction pathways are heterotrimeric guanine nucleotide binding proteins (G proteins) coupled to cell surface receptors (for review see refs 1-4 and 72). G proteins relay signals generated by various stimuli including photons, odors, and various hormones and neurotransmitters. Defects in G protein activity can cause a variety of diseases. G proteins exist as heterotrimeric complexes of α, β, and γ subu...

Examples

Embodiment 1

[0068] G protein, heterotrimeric guanine nucleotide-binding protein; RGS4, regulator of G protein signaling; G iα1 , the Gα subunit of the heterotrimeric G protein, G iα1 -AIF 4 - , with Mg 2+ , GDP and AlF 4- Complex Gα subunit of heterotrimeric G protein stabilized in GTP hydrolysis transition state, DTT, DL-1,4-dithiothreitol; GTP, guanosine triphosphate; GDP, guanosine diphosphate; NMR, nuclear magnetic resonance; 2D, two-dimensional; 3D, three-dimensional; HSQC, heteronuclear single-level quantum coherence spectroscopy; HMQC, heteronuclear multilevel quantum coherence spectroscopy; TPPI, time-proportional phase increment; Nuclear Overhauser effect; NOESY, nuclear Overhauser enhanced spectroscopy; COZY, correlation spectroscopy; HNHA, amide proton to nitrogen to CαH proton correlation (amide proton to nitrogen to CαH proton HNHB, amide proton to nitrogen to CβH proton correlation (amide proton to nitrogen to CβHproton correlation); CT-HCACO, constant time CαH proton t...

Embodiment 2

[0075] The secondary structure of the RGS4-core (outlined in Figure 1) was obtained from 15 N-modified NOESY-HMQC and 13 C - Characteristic NOE data of NH, Hα and Hβ protons of the corrected NOESY-HMQC spectrum, obtained from HNHA 3J HNα coupling constants, slowly exchanging NH protons and 13 Cα and 13 Cβ secondary chemical shifts (for review see: (56) and (78)). RGS4-core solution NMR has been determined to consist of seven helical regions corresponding to the following residues: residues 7-12 (α1); 17-36 (α2); 40-53 (α3); 61-71 (α4); 86-95 (α5); 105-125 (α6) and 128-132 (α7). The RGS4-core overall fold essentially consists of two 4-helical bundles, both of which have a long helical region α6 portion. A clear difference between the RGS4 core secondary structure in solution and the X-ray structure of the RGS4-Giα1 complex was unexpectedly observed at the C-terminus. The X-ray structure shows that residues 104-116 and 119-129 are helical when looking at residues V5-T132. ...

Embodiment 3

[0088] RGS4-G iαi The complex (AGR1) X-ray structure was read into SYBYL (Tripos) and all substructures except strand E (RGS4) were deleted. Also, remove all water. Polar hydrogen was added and Kollman United Atom force field optimization was used. All remaining hydrogen was then added. MOLCAD was then used to generate a surface of all residues believed to be involved in T182 (Ga-binding site) binding. These RGS4-core residues include: I21, I27, F30, F33, L34, E37, S39, N42, I43, W46, I110, L113, M114, D117, S118, R121. The surface area is calculated based on MOLCAD surfaces. The surface area of ​​the same residues in the free RGS4 NMR structure was also calculated using MOLCAD. The calculated surface area of ​​the free RGS4 NMR structure is 404.56 Å 2 . The calculated surface area of ​​the crystal structure is 321.88 Å 2 . The difference in surface area is 82.67 Å 2 , that is about a 20% change in surface area between the two structures. The surface area of ​​the M...

Background of the invention

[0001] A wide variety of biochemical processes, especially those involving protein-protein interactions, are thought to be mediated by induced conformational changes in protein targets. The resulting structural changes in a protein can then be used to explain changes in its function (eg enzymatic activity) or its affinity for another protein in a biological system. Conformational changes have been proposed to occur in eukaryotic cells in cascades associated with steps in certain signal transduction pathways. Ubiquitous components of such signal transduction pathways are heterotrimeric guanine nucleotide binding proteins (G proteins) coupled to cell surface receptors (for review see refs 1-4 and 72). G proteins relay signals generated by various stimuli including photons, odors, and various hormones and neurotransmitters. Defects in G protein activity can cause a variety of diseases. G proteins exist as heterotrimeric complexes of α, β, and γ subu...

Examples

Embodiment 1

[0068] G protein, heterotrimeric guanine nucleotide-binding protein; RGS4, regulator of G protein signaling; G iα1 , the Gα subunit of the heterotrimeric G protein, G iα1 -AIF 4 - , with Mg 2+ , GDP and AlF 4- Complex Gα subunit of heterotrimeric G protein stabilized in GTP hydrolysis transition state, DTT, DL-1,4-dithiothreitol; GTP, guanosine triphosphate; GDP, guanosine diphosphate; NMR, nuclear magnetic resonance; 2D, two-dimensional; 3D, three-dimensional; HSQC, heteronuclear single-level quantum coherence spectroscopy; HMQC, heteronuclear multilevel quantum coherence spectroscopy; TPPI, time-proportional phase increment; Nuclear Overhauser effect; NOESY, nuclear Overhauser enhanced spectroscopy; COZY, correlation spectroscopy; HNHA, amide proton to nitrogen to CαH proton correlation (amide proton to nitrogen to CαH proton HNHB, amide proton to nitrogen to CβH proton correlation (amide proton to nitrogen to CβHproton correlation); CT-HCACO, constant time CαH proton t...

Embodiment 2

[0075] The secondary structure of the RGS4-core (outlined in Figure 1) was obtained from 15 N-modified NOESY-HMQC and 13 C - Characteristic NOE data of NH, Hα and Hβ protons of the corrected NOESY-HMQC spectrum, obtained from HNHA 3J HNα coupling constants, slowly exchanging NH protons and 13 Cα and 13 Cβ secondary chemical shifts (for review see: (56) and (78)). RGS4-core solution NMR has been determined to consist of seven helical regions corresponding to the following residues: residues 7-12 (α1); 17-36 (α2); 40-53 (α3); 61-71 (α4); 86-95 (α5); 105-125 (α6) and 128-132 (α7). The RGS4-core overall fold essentially consists of two 4-helical bundles, both of which have a long helical region α6 portion. A clear difference between the RGS4 core secondary structure in solution and the X-ray structure of the RGS4-Giα1 complex was unexpectedly observed at the C-terminus. The X-ray structure shows that residues 104-116 and 119-129 are helical when looking at residues V5-T132. ...

Embodiment 3

[0088] RGS4-G iαi The complex (AGR1) X-ray structure was read into SYBYL (Tripos) and all substructures except strand E (RGS4) were deleted. Also, remove all water. Polar hydrogen was added and Kollman United Atom force field optimization was used. All remaining hydrogen was then added. MOLCAD was then used to generate a surface of all residues believed to be involved in T182 (Ga-binding site) binding. These RGS4-core residues include: I21, I27, F30, F33, L34, E37, S39, N42, I43, W46, I110, L113, M114, D117, S118, R121. The surface area is calculated based on MOLCAD surfaces. The surface area of ​​the same residues in the free RGS4 NMR structure was also calculated using MOLCAD. The calculated surface area of ​​the free RGS4 NMR structure is 404.56 Å 2 . The calculated surface area of ​​the crystal structure is 321.88 Å 2 . The difference in surface area is 82.67 Å 2 , that is about a 20% change in surface area between the two structures. The surface area of ​​the M...

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