Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
2000-09-18
2004-01-27
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S325000, C435S348000, C435S320100, C536S023100, C536S023200
Reexamination Certificate
active
06682921
ABSTRACT:
1. INTRODUCTION
The present invention concerns crystalline forms of polypeptides corresponding to the catalytic domain of receptor tyrosine kinases of the non-insulin receptor type. Such tyrosine kinases include receptors of a class that are not covalently cross-linked but are understood to undergo ligand-induced dimerization (such as the FGF-receptor), as well as cytoplasmic tyrosine-kinases. The invention also concerns methods for obtaining such crystals and to the high-resolution X-ray diffraction structures and atomic structure coordinates obtained therefrom. The crystals of the invention, and the atomic structure coordinates obtained therefrom, are useful for solving the crystal and solution structures of the tyrosine kinase domains and for identifying compounds that bind to domains of receptor and non-receptor tyrosine kinases.
2. BACKGROUND OF THE INVENTION
Growth factors play important roles in the control of cell growth, differentiation, metabolism and oncogenesis. The signals generated by a growth factor are transduced across the cellular membrane by transmembrane receptors specific for the growth factor. The diverse biological effects of growth factors are mediated by a large family of cell surface transmembrane receptors with intrinsic protein tyrosine kinase (PTK) activity. The extracellular portion of receptor PTKs contain the binding site for its particular growth factor/ligand, whereas the tyrosine kinase activity resides in the cytoplasmic portion. Binding of a growth factor to the extracellular domain of this receptor results in autophosphorylation of specific tyrosine residues in the cytoplasmic domain. These phosphotyrosines either stimulate PTK activity or serve as binding sites for downstream signalling proteins containing Src-homology 2 (SH2) or phosphotyrosine binding (PTB) domains.
Eighteen classes or subfamilies of human receptor PTKs have been identified to date, including the insulin-receptor (IR), EGF-receptor, PDGF receptor and FGF-receptor. Ligand-induced dimerization of receptors such as the EGF, PDGF and FGF receptors is thought to be essential for activation. Growth factors, such as PDGF are dimeric molecules which, by themselves, are able to induce PDGF-receptor dimerization. However, FGFs are monomeric and are unable, by themselves; to induce receptor dimerization. Dimerization of FGF receptors is thought to be mediated by FGF in concert with heparin sulfate proteoglycans (soluble or cell surface bound).
In contrast to the EGF, PDGF and FGF receptors, which are monomeric and dimerize upon ligand binding, the insulin receptor exists as a “dimer.” In fact, the insulin receptor is a disulphide-linked &agr;
2
&bgr;
2
heterotetramer. Binding of insulin to the extracellular &agr;-chains is thought to cause a change within the quaternary structure of the receptor that results in autophosphorylation of specific tyrosines in the cytoplasmic portion of the &bgr; chains.
In an effort to elucidate the mechanisms underlying kinase activation, the crystal structure of such proteins is often sought to be determined. The crystal structures of several protein serine/threonine kinases have been reported: cyclic-AMP-dependent protein kinase (CAPK; Knighton et al., 1994); cyclin-dependent kinase 2 (CDK2; DeBondt et al., 1993); mitogen-activated protein kinase (MAPK; Zhang et al., 1994); and twitchin kinase (Hu et al., 1994). However, the crystalline structure of only one receptor tyrosine kinase has been determined—the unphosphorylated apo form of the tyrosine kinase domain of the insulin receptor (Hubbard et al., 1994).
Despite these reports, the ability to obtain crystalline forms of the tyrosine kinase domains of non-insulin receptor tyrosine kinases; i.e., cytoplasmic tyrosine kinases and/or receptor tyrosine kinases that undergo ligand-mediated dimerization, has not been realized. A particularly illuminating example is the EGF receptor; to the Applicant's knowledge, researchers armed with the knowledge of how to obtain crystals of the tyrosine kinase domains of both the insulin receptor and serine/threonine kinases have attempted to obtain crystals of the tyrosine kinase domain of EGF receptor without success.
3. SUMMARY OF THE INVENTION
The invention relates to crystalline forms of polypeptides corresponding to the catalytic domains of receptor tyrosine kinases of the non-insulin receptor type. Such tyrosine kinases include receptors that are not covalently cross-linked, but are believed to undergo ligand-induced dimerization, as well as cytoplasmic tyrosine kinases. The polypeptides of the invention include, but are not limited to, crystallized polypeptides corresponding to the native or mutated catalytic domain of tyrosine kinases (ie., the non-insulin receptor-type described above), derivative crystals (i.e., heavy atom derivatives), and co-crystals of the native or mutated catalytic domain in association with one or more compounds, including but not limited to cofactors, substrates, substrate analogs, inhibitors, allosteric effectors, etc., and preferably compounds that bind the catalytic site.
Preferably, the crystalline catalytic domains of the invention are of sufficient quality to provide for a determination of the three-dimensional X-ray diffraction structure of the crystalline polypeptide to a resolution of about 1.5 Å to about 2.5 Å.
The invention is based, in part, on the Applicants' discovery and elucidation of the sequence requirements for the successful crystallization of polypeptides corresponding to catalytic domains of receptor tyrosine kinases that are not covalently cross-linked and are believed to undergo ligand-induced dimerization—a goal which heretofore remained elusive. In this regard, the Applicants have determined that at least about 20 amino acid residues (+/−5 amino acid residues) upstream of the first glycine in the conserved glycine-rich region of the catalytic domain, and at least about 17 amino acid residues (+/−5 amino acid residues) downstream of the conserved arginine located at the C-terminal boundary of the catalytic domain are required to engineer a polypeptide suitable for crystallization.
In those cases where the resulting polypeptide contains cysteine residues that interfere with crystallization, such cysteine residues can be substituted with an appropriate amino acid that does not readily form covalent bonds with other amino acid residues under crystallization conditions, e.g., such substitutions include, but are not limited to Ala, Ser, or Gly. Any cysteines located in a non-helical or non-&bgr;-strand segment based on secondary structural assignments are candidates for replacement. Cysteines located in domains corresponding to the glycine-rich loop, the kinase insert, the juxtamembrane region or the activation loop are prime candidates for replacement. However, substitutions of cysteine residues that are conserved among the kinases should be avoided (e.g., substitutions of the highly conserved cysteine residues located at the C-terminus, positions 725 and 736 in
FIG. 6A
, should be avoided).
The invention is demonstrated by way of example, for the fibroblast growth factor (FGF) receptor-1 (FGF-R1). The examples demonstrate that the crystal structure of the tyrosine kinase domain of the FGF-R1 has been determined to 2.0 Å resolution; the crystal structure of the FGF-R1 catalytic domain in complex with an ATP analog is described to 2.3 Å resolution.
The crystalline catalytic domains are useful for elucidating the mechanism by which the receptor tyrosine kinases are activated by ligand-induced dimerization, and for the identification of compounds that bind to the catalytic domain.
3.1 Definitions
As used herein, the following terms shall have the following meanings:
“Native Tyrosine Kinase Domain or Native Catalytic Domain:” As used herein, “native tyrosine kinase domain” or “native catalytic domain” refers to that portion or domain of a naturally occurring cytoplasmic tyrosine kinase or non-insulin receptor tyrosine kinase which possesses protein tyrosine
Hubbard Stevan R.
Mohammadi Moosa
Schlessinger Joseph
Burrous Beth A.
Foley & Lardner
Nashed Nashaat T.
New York University
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