WO 2011/132001 A1 - Crystal Structure - The Lens - Free & Open Patent and Scholarly Search

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WO 2011/132001 A1

Crystal Structure

Published: Oct 27, 2011
Earliest Priority: Apr 22 2010
Family: 1
Cited Works: 28
Cited by: 0
Cites: 1
Sequences: 9
Additional Info: Cited Works Full text Published Sequence

Abstract

A crystal comprising folliculin (FLCN) C-terminus having a space group C 2 2 21 with unit cell dimensions of a=86.1 Å, b=100.0 Å, c=107.6 Å is provided. A crystal comprising folliculin (FLCN) C-terminus and GDP having a space group C2 with unit cell dimensions of a=98.90 Å, b=85.41 Å, c=65.85 Å is also provided. Also provided is a method of crystallizing folliculin C-terminus and GDP and methods of rational drug design.

Claims

A crystal comprising folliculin (FLCN) C-terminus having a space group C 2 2 21 with unit cell dimensions of a=86.1 A, b=100.0 A, c=107.6 A, more preferably with unit cell dimensions of a=86.050 A, b=99.951 A, c=107.584 A.
A crystal comprising folliculin (FLCN) C-terminus wherein said crystal effectively diffracts X-rays for the determination of the atomic co-ordinates to a resolution greater than or substantially equal to 1.9 A.
A crystal comprising folliculin (FLCN) C-terminus wherein said crystal effectively diffracts X-rays for the determination of the atomic co-ordinates to a resolution of about 1.9 A.
A crystal comprising folliculin (FLCN) C-terminus having the atomic coordinates defined by the coordinates of Table 3, 3A, or 3B.
A crystal according to any preceding claim wherein the folliculin (FLCN) C- terminus carries at least one mutation when compared to the amino acid sequence of wild type folliculin (FLCN) C-terminus.
A crystal according to any preceding claim wherein the folliculin (FLCN) C- terminus is a triple cysteine mutant.
A crystal according to claim 6 wherein the folliculin (FLCN) C-terminus triple cysteine mutant comprises the amino acid sequence set out in SEQ ID NO: 4.
A crystal according to any of claims 1 and 4 to 7 wherein said crystal effectively diffracts X-rays for the determination of the atomic co-ordinates to a resolution greater than or substantially equal to 1.9 A.
A crystal according to any of claims 1 and 4 to 7 wherein said crystal effectively diffracts X-rays for the determination of the atomic co-ordinates to a resolution of about 1.9 A.
A crystal according to any of claims 1 to 3 and 5 to 9 having the atomic coordinates defined by the coordinates of Table 3, 3A or 3B.
1 1. A crystal comprising folliculin (FLCN) C-terminus and GDP having a space group C2 with unit cell dimensions of a=98.9 A, b=85.4 A, c=65.9, more preferably with unit cell dimensions A a=98.90 A, b=85.41 A, c=65.85 A.
A crystal comprising folliculin (FLCN) C-terminus and GDP wherein said crystal effectively diffracts X-rays for the determination of the atomic co- ordinates to a resolution greater than or substantially equal to 2.7 A.
A crystal comprising folliculin (FLCN) C-terminus and GDP wherein said crystal effectively diffracts X-rays for the determination of the atomic coordinates to a resolution of about 2.7 A.
1 . A crystal comprising folliculin (FLCN) C-terminus and GDP having the atomic coordinates defined by the coordinates of Table 1 or 1 A.
A crystal according to any of claims 11 to 14 wherein the folliculin (FLCN) C- terminus carries at least one mutation when compared to the amino acid sequence of wild type folliculin (FLCN) C-terminus.
A crystal according to any of claims 11 to 15 wherein the folliculin (FLCN) C- terminus is a triple cysteine mutant of folliculin (FLCN) C-terminus.
A crystal according to any of claims 11 to 16 wherein the folliculin (FLCN) C- terminus comprises the amino acid sequence set out in SEQ ID NO: 4.
A crystal according to any of claims 11 or 14 to 18 wherein said crystal effectively diffracts X-rays for the determination of the atomic co-ordinates to a resolution of about 2.7 A.
A crystal according to any of claims 11 or 14 to 17 wherein said crystal effectively diffracts X-rays for the determination of the atomic co-ordinates to a resolution greater than or substantially equal to 2.7 A.
A crystal according to any of claims 1 1 to 13 or 15 to 19 having the atomic coordinates defined by the coordinates of Table 1 or 1A.
A crystal comprising folliculin (FLCN) C-terminus wherein the folliculin (FLCN) C-terminus comprises the amino acid sequence set out in SEQ ID No. 4.
A crystal according to claim 21 further comprising GDP.
A method of crystallizing folliculin (FLCN) C-terminus comprising providing folliculin (FLCN) C-terminus and crystallizing the folliculin (FLCN) C-terminus.
A method according to claim 23 further comprising mutating the sequence of folliculin FLCN C-terminus to introduce at least one mutation before crystallizing the folliculin (FLCN) C-terminus.
A method according to claims 23 or 24 wherein the folliculin (FLCN) C- terminus cysteine residues 454, 503 and 506 are mutated to alanine residues.
A method of crystallizing folliculin C-terminus and GDP comprising
(i) providing folliculin (FLCN) C-terminus
(ii) mutating folliculin (FLCN) C-terminus cysteine residues 454, 503 and 506 to alanine residues
(iii) preparing a protein solution comprising triple cysteine mutant folliculin (FLCN) C-terminus
(iv) mixing said protein solution with a solution comprising GTP
(v) subjecting the composition to conditions which promote crystallization.
A method according to any of claims 23 to 26 wherein the crystallization occurs from a mother liquor comprising about 0.2M LiS04, about 0.1 M Bis-Tris pH
5 and about 25% PEG 3350.
A method according to any of claims 23 to 27 further comprising harvesting the crystals in cryo-protectant solution comprising about 30% polypropylene glycol.
A protein comprising the amino acid sequence as set out in SEQ ID NO: 4.
An expression vector comprising a nucleotide sequence which encodes the amino acid sequence as set out in SEQ ID NO: 4 or a portion of the amino acid sequence as set out in SEQ ID NO: 4.
Use of a protein comprising the amino acid sequence as set out in SEQ ID NO: 4 or a portion thereof for protein crystallography.
A computer-based method of rational drug design which comprises: providing the structure of folliculin (FLCN) C-terminus having the structure defined by the coordinates of Table 1 , Table 1 A, Table 3, Table 3A or Table 3B, or a portion thereof; providing the structure of a candidate molecule; and fitting the structure of the candidate to the structure of folliculin (FLCN) C-terminus having the structure defined by the coordinates of Table 1 , Table 1 A, Table 3, Table 3A or Table 3B, or a portion thereof.
A computer-based method of rational drug design according to claim 32 wherein the candidate molecule interacts with the nucleotide-binding motif of folliculin (FLCN) C-terminus.
A computer-based method of rational drug design according to claim 33 wherein the nucleotide-binding motif of folliculin (FLCN) C-terminus is the GTP binding motif of folliculin (FLCN) C-terminus. 35. A computer-based method of rational drug design according to any of claims 32 to 34 wherein the candidate molecule interacts with the NKIE motif of folliculin (FLCN) C-terminus.
A computer-based method of identifying molecules that can bind to folliculin (FLCN) C-terminus, the method comprising: providing the structure of folliculin (FLCN) C-terminus having the structure defined by all or a portion of the coordinates of Table 1 , Table 1 A, Table 3, Table 3A or Table 3B; providing the structure of a candidate molecule and comparing the coordinates of the candidate molecule against those of folliculin (FLCN) C-terminus to identify whether the candidate molecule can bind to folliculin (FLCN) C-terminus.
A computer-based method according to claim 36 wherein the coordinates of the candidate molecule are compared against those of folliculin (FLCN) C-terminus to identify whether the candidate molecule can bind to the nucleotide-binding motif of folliculin (FLCN) C-terminus.
A computer-based method according to claim 37 wherein the nucleotide- binding motif is the GTP binding motif of folliculin (FLCN) C-terminus.
A computer-based method according to claim 37 or 38 wherein the nucleotide-binding motif is the NKIE binding motif of folliculin (FLCN) C-terminus.
A method for providing a molecule capable of interacting with a folliculin (FLCN) C-terminus structure, the method comprising:
(i) providing the structure of folliculin (FLCN) C-terminus or a portion of folliculin (FLCN) C-terminus, said structure being defined by all or a portion of the coordinates of Table 1 , Table 1 A, Table 3, Table 3A or Table 3B, with a root mean square deviation of not more than 2.7 A providing a structure of a molecule to be fitted to said folliculin (FLCN) C-terminus structure or selected portion thereof
(iii) fitting said structure of a molecule to said folliculin (FLCN) C-terminus structure or selected portion thereof and
(iv) obtaining or synthesising a compound which has the structure of said molecule.
A method according to claim 40 further comprising the step of contacting the molecule with the folliculin (FLCN) C-terminus in order to determine the ability of the molecule to interact with folliculin (FLCN) C-terminus.
A method according to claim 40 or 41 wherein the portion of coordinates includes the coordinates of atoms from one or more of residues Asn484, Lys485, Ile486 and Glu487.
A method of determining the structure of a compound capable of binding to folliculin (FLCN) C-terminus comprising
(i) providing a sample comprising folliculin (FLCN) C-terminus
(ii) providing a sample of a compound
(iii) mixing said sample comprising folliculin (FLCN) C-terminus with said sample of a compound
(iv) crystallizing the mixture and
(v) determining the crystal structure of the mixture.
A method according to claim 43 wherein the folliculin (FLCN) C-terminus carries at least one mutation when compared to the amino acid sequence of wild type folliculin (FLCN) C-terminus.
A method according to claim 43 or 44 wherein the crystal structure of the mixture is determined using the data from Table 1 , Table 1 A, Table 3, Table 3A or Table 3B, or a portion thereof.
A method of identifying the structure of a compound capable of binding to folliculin (FLCN) C-terminus comprising
(i) providing a crystal comprising folliculin (FLCN) C-terminus
(ii) soaking the crystal with the compound to form a complex (iii) determining the structure of the complex using the data from
Table 1 , Table 1 A, Table 3, Table 3A or Table 3B, or a portion thereof.
A method for determining the structure of a protein said method comprising:
(i) providing the coordinates of Table 1 , Table 1 A, Table 3, Table 3A or Table 3B, or selected coordinates thereof and either (a) positioning said coordinates in the crystal unit cell of said protein or (b) obtaining NM spectra of said protein and assigning NMR spectra peaks of said protein by manipulating said co-ordinates.
A method for determining the structure of a complex comprising a protein and a compound capable of interacting with said protein, said method comprising:
(i) providing the coordinates of Table 1 , Table 1 A, Table 3, Table 3A or Table 3B, or selected coordinates thereof
(ii) obtaining NMR spectra of said complex
(ii) assigning NMR spectra peaks of said complex by manipulating said co-ordinates
A crystal, a method of crystallizing, a computer-based method, a protein, an expression vector, use of a protein, a computer-based method of rational drug design, a computer-based method of identifying molecules, a method for providing a molecule, a method of determining the structure of a compound, protein or complex or a method of identifying the structure of a compound as herein described and illustrated.

Applicants

Myrovlytis Technology Ventures Ltd
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Nookala Ravi
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Inventors

Nookala Ravi
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CPC Classifications

C30B29/58
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C07K14/47
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C07K2299/00
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C30B7/04
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IPC Classifications

C30B7/04
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C30B29/58
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Document History

Publication: Oct 27, 2011
WO 2011/132001 A1
Application: Apr 26, 2011
GB 2011050819 W
Priority: Dec 17, 2010
GB 201021429 A
Priority: Apr 22, 2010
GB 201006722 A

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