Three dimensional structure of a ZAP tyrosine protein kinase...

Details Three dimensional structure of a ZAP tyrosine protein kinase... Three dimensional structure of a ZAP tyrosine protein kinase...

1997-11-18

2001-06-26

Nashed, Nashaat T. (Department: 1652)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving transferase

C435S194000, C436S086000, C436S169000, C702S019000

Reexamination Certificate

active

06251620

ABSTRACT:

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A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
FIELD OF THE INVENTION
The invention relates to human ZAP-70, and in particular, to the region of ZAP-70 containing the tandem Src homology-2 (“SH2”) domains, to crystalline forms thereof, liganded or unliganded, which are particularly useful for the determination of the three-dimensional structure of the protein. The three dimensional structure of the tandem SH2 region of ZAP provides information useful for the design of pharmaceutical compositions which inhibit the biological function of ZAP and other proteins of the ZAP family, particularly those biological functions mediated by molecular interactions involving one or both SH2 domains.
BACKGROUND
Safe and effective immunosuppressive agents are required for the treatment of patients suffering from autoimmune disorders and for recipients of transplanted organs or tissues. For instance, in the absence of an effective immunosuppressive agent, patients often reject a transplanted organ, sometimes with fatal consequences. The immunosuppressive agent must block the immune response, but must also be sufficiently well tolerated by the body to permit chronic application. For instance, one compound with immunosuppressive activity, FK506, has been used to prevent rejection of transplanted livers. However, severe kidney toxicity has been observed in patients receiving FK506, in some cases requiring kidney transplant following the liver transplant.
Research aimed at discovering new immunosuppressive agents has been hampered by the lack of information about precise molecular mechanisms of the immune response. As a result, random screening of compounds has accounted for a substantial share of research efforts aimed at identifying new immunosuppressive drugs. More recently, “structure-based” approaches to drug design have been attempted. For example, compounds designed to bind to the protein FKBP, one of the cellular targets of FK506, were synthesized as candidate immunosuppressive agents. Those efforts were unfortunately doomed by the lack of understanding of the actual molecular mechanism of immunosuppression mediated by FK506. It is now known that FKS506 binds in a complex with two proteins, FKBP and calcineurin. FK506's immunosuppressive effects are due to the inhibition of calcineurin in T cells. However, since calcineurin is present and important in other cells, FK506 affects other cells and tissues leading to undesired effects.
Meanwhile, independent efforts have led to the identification of a protein tyrosine kinase, ZAP-70, as a critical mediator of the immune response. Blocking the biological function of ZAP-70 will lead to immunosuppression. Unfortunately, until now, three-dimensional structural details of ZAP-70 have been completely unknown. In the absence of three-dimensional structural details for that protein, designing inhibitors based on that structure would have been impossible. We have now obtained crystals of a critical region of ZAP-70 containing its tandem SH2 domains, with and without bound ligands of various types, and have determined its three dimensional structure. With this information, it is now possible for the first time to rationally design inhibitors of ZAP-70 which can function as immunosuppressive agents, e.g. compounds which inhibit molecular interactions involving one or both of the ZAP-70 SH2 domains. Although the three-dimensional structures for several individual SH2 domains of other proteins are known, no one has heretofore reported determining the three-dimensional structure of a tandem SH2 region. And, as we discuss below, the three-dimensional coordinates of previously known SH2 domains would have been insufficient to solve the structure of the ZAP-70 tandem SH2 region.
SUMMARY OF THE INVENTION
This invention concerns the region of human ZAP-70 spanning its two SH2 domains. We refer to that region as the “ZAP tandem SH2 region” or simply “ZAP-NC” (SEQ ID NO: 38), since the region contains both the more
N
-terminal SH2 domain and the more
C
-terminal SH2 domain of human ZAP-70 (see FIG.
4
). The invention begins with obtaining crystals of human ZAP-NC (SEQ ID NO: 36), complexed or uncomplexed with various ligands, of sufficient quality to determine the three dimensional (tertiary) structure of the protein by X-ray diffraction methods.
In considering our work, it should be appreciated that obtaining protein crystals in any case is a somewhat unpredictable art, especially in cases in which the practitioner lacks the guidance of prior successes in preparing and/or crystallizing any closely related proteins. Obtaining our first crystals of ZAP-NC (SEQ ID NO: 36) was therefore itself an unexpected result. In addition, our determination of the three-dimensional structure of ZAP-NC (SEQ ID NO: 36) represents the first solution of a three-dimensional structure for a tandem SH2 region from any protein and revealed an unpredicted array of surface features which contained truly surprising structural aspects. Our results are useful in a number of applications.
For example, the knowledge obtained concerning ZAP-NC (SEQ ID NO: 36) can be used to model the tertiary structure of related proteins. For instance, the structure of renin has been modeled using the tertiary structure of endothiapepsin as a starting point for the derivation. Model building of cercarial elastase and tophozoite cysteine protease were each built from known serine and cysteine proteases that have less than 35% sequence identity. The resultant models were used to design inhibitors in the low micromolar range. (
Proc. Natl. Acad. Sci.
1993, 90, 3583). Furthermore, alternative methods of tertiary structure determination that do not rely on X-ray diffraction techniques and thus do not require crystallization of the protein, such as NMR techniques, are simplified if a model of the structure is available for refinement using the additional data gathered by the alternative technique. Thus, knowledge of the tertiary structure of the ZAP tandem SH2 region provides a significant window to the structure of the other ZAP family members, including for example SYK.
Knowledge of the three-dimensional structure of a tandem SH2 region such as ZAP-NC (SEQ ID NO: 36) provides a means for investigating the mechanism of action of the protein and tools for identifying inhibitors of its function. For example, SH2 domains are known to be involved in intramolecular and intermolecular interactions, usually protein-protein interactions, which are critical for biological activity of the SH2-bearing protein. Knowledge of the three-dimensional structure of the tandem SH2 region allows one to design molecules capable of binding thereto, including molecules which are thereby capable of inhibiting the interaction of the tandem SH2 region with its natural ligand(s).
Accordingly, one object of this invention is to provide a composition comprising a protein in crystalline form having a peptide sequence derived or selected from that of a protein of the ZAP family. The protein will comprise at least one, and preferably two SH2 domains, e.g., a protein containing the tandem SH2 region of ZAP-70, SYK or other related tandem SH2 containing protein. In the case of ZAP-70, the protein may comprise a peptide sequence spanning at least amino acid residues 3-279. Such a crystalline composition may contain one or more heavy atoms, e.g., one or more lead, mercury, gold and/or selenium atoms, for instance. Such a heavy atom derivative may be obtained, for example, by expressing a gene encoding the protein under conditions permitting the incorporation of one or more heavy atom labels (e.g. as in the incorporation of selenomethionine), reacting the protein with a reagent capable of linking a heavy atom to the

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