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Team Of Max Planck Scientists Elucidates Structure Of A Protein Segment Involved In 'Von Recklinghausen Neurofibromatosis'

Ribbon diagram of a hypothetical complex between Ras and NF1-GAP. The figure was generated by superimposing the structural model of NF1-GAP with that of the complex between Ras and the GAP segment of p120GAP, to give a realistic albeit hypothetical impression of how NF1-GAP interacts with Ras. Regions of Ras important for the interactions are highlighted in turquoise, those of NF1 in light blue. The large domain of NF1-GAP is in blue, the small one in yellow. Segments that are presumably mobile in the protein are in red, shown in conformations that are derived from those of the corresponding regions in p120GAP. The position of the functionally most important residues are highlighted as balls in magenta on NF1-GAP and in green on Ras. Positions of mutations found in NF1 patients are highlighted as white balls. Full size image available through contact

Scientists at the Max Planck Institute for Molecular Physiology in Dortmund and of the Max Planck Institute for Medical Research in Heidelberg (Germany) have determined the first structure of a functional protein unit that is involved in the development of ‘von Recklinghausen neurofibromatosis’. This unit is a regulator of another protein involved in the pathogenesis of numerous tumors, linking neurofibromatosis to tumor development. The work, published in the August 3, 1998, issue of EMBO Journal, confirms mechanistic ideas about the function of this protein stemming from the authors' fundamental structural and biochemical studies of the related protein p120GAP (Mittal et al., 1996, Science 273, 115-117; Scheffzek et al., 1996, Nature 384, 591-596; Scheffzek et al., 1997, Science 277, 333-338; Ahmadian et al., 1997, Nature Struct. Biol. 4, 686-689).

von Recklinghausen neurofibromatosis, also called type 1 neurofibromatosis or simply NF1, is an inherited disease that predisposes the formation of certain types of tumors and may be associated with a number of other symptoms including learning disabilities and mental retardation. So-called Lish nodules of the iris or pigmentation abnormalities of the skin (so-called ‘café-au-lait spots’) and neurofibromas, benign cutaneous tumors, are hallmarks of NF1 with important diagnostic relevance. In rare cases, NF1 may develop into malignant tumors.

The so-called NF1 gene is responsible for the development of the disease. It codes a huge protein, termed neurofibromin, comprising nearly 3000 amino acids, and it is disrupted or mutated in patients affected with NF1. A segment comprising ca. 10% of the whole protein represents the only known functional module of neurofibromin. It functions as a negative regulator of Ras, another protein that is intimately involved in the regulation of cell growth and differentiation. The physiological importance of this is underscored by the observation that Ras carries characteristic mutations in 30% of all cancer tumors.

Ras can be thought of as a binary signal switch cycling between ON and OFF states which are characterized in terms of a small molecule, a guanine nucleotide, bound to the protein. In the resting cell, Ras is tightly bound to guanosine diphosphate (GDP), which is exchanged for guanosine triphosphate (GTP) upon binding of extracellular stimuli to cell membrane receptors. In the GTP-bound form, Ras interacts specifically with so-called effector proteins, thereby initiating cascades of protein-protein interactions that may finally lead to cell proliferation. To return to the inactive OFF state, Ras cleaves off the terminal phosphate moiety, the g -phosphate, of GTP in an enzymatic process, the intrinsic GTPase reaction. The remaining GDP-bound Ras is no longer able to interact with effectors, it is switched OFF. The process of GTP cleavage is very slow: Ras splits one GTP every 30 minutes. But upon interaction with neurofibromin, the rate is enhanced 100,000 fold: the underlying process is termed GTPase activation, making neurofibromin a GTPase activating protein (GAP). A common feature of the Ras mutations found in tumors is the inability of the resulting Ras proteins to cleave GTP efficiently, i.e. to turn off the switch; in addition they are not sensitive to GTPase activation by neurofibromin. Thus, understanding neurofibromin function also means understanding aspects of Ras function, linking NF1 to cancerogenesis.

Apart from neurofibromin, there are other GTPase activating proteins specific for Ras; in living organisms, it is indeed common that identical processes are carried out by different components. The first GAP to be discovered was the p120GAP (Trahey & McCormick, 1987, Science 238, 542-545). In fundamental studies involving biochemical and structural methods, the research team has previously included p120GAP to elucidate the molecular mechanism behind the GTPase activation process. p120GAP, represented by the segment of the protein that is sufficient to stimulate Ras-mediated GTP hydrolysis, complements the active site which is where the reaction takes place. It does so by two strategies: Firstly, it supplies an amino acid (an arginine) that participates directly in the reaction; its mutation to another amino acid destroys GAP activity. Secondly, it stabilizes the functionally most important amino acids on Ras, thereby aligning the catalytic machinery (Scheffzek et al., Science 277, 333-338).

The structure of neurofibromin GAP resembles the structure of the corresponding segment of p120GAP: it is an elongated molecule that is composed of a small and a large domain, the latter of which contains all the functionally important residues. Together with the biochemical analyses, the structural similarity confirms that it functions by the same mechanism. On the basis of the structural work on neurofibromin and p120GAP complexed with Ras, the effect of mutations in the GAP segment, as found in NF1-patients, can be analyzed; e.g. the catalytic arginine contributed by GAP has been found mutated in an NF1 patient; the resulting GAP protein has an intact structure but is completely inactive (Klose et al., 1998, Hum. Mol. Genet. 7, 1261-1268). Since this mutation was the only one found in the whole NF1 gene of the respective patient, it appears that loss of GAP function is sufficient for the development of neurofibromatosis.

The medical impact of the work on neurofibromin and p120GAP is twofold: on the one hand it is now possible to picture how neurofibromin acts on Ras and to analyze mutations in the light of the structure; on the other hand, the structure of the contact area between GAP and Ras suggests that it might be attractive to search for small molecules that may act as surrogate GAPs in tumor cells where Ras is fatally mutated or its GTP-bound levels are increased for other reasons, e.g. due to a missing neurofibromin function. The 3-dimensional picture of GAP and of its communication with Ras may suggest how to proceed, but also indicates us that it will be an extremely formidable task.

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