Structural differences in the minimal catalytic domains of the GTPase-activating proteins p120GAP and neurofibromin.
The kinetic properties for the enzymatic stimulation of the GTPase reaction of p21(ras) by the two GTPase-activating proteins (GAPs) p120(GAP) and neurofibromin are different. In order to understand these differences and since crystallization attempts have only been successful with truncated fragments, structure/function requirements of the catalytic core of these proteins were investigated. Differences in size of the minimal catalytic domains of these two proteins were found as determined by limited proteolysis. The minimal catalytic domain has a molecular mass of 30 kDa in the case of p120(GAP) and of 26 kDa in the case of neurofibromin. Both catalytic domains contain the homology boxes as well as the residues perfectly conserved among all Ras GAPs. The C termini of these fragments are identical, whereas the N-terminal part of the minimal p120(GAP) domain is 47 amino acids longer. These newly identified minimal catalytic fragments were as active in stimulating GTPase activity toward p21(ras) as the corresponding larger fragments GAP-334 and NF1-333 from which they had been generated via proteolytic digestion. Recently it was postulated that a fragment of 91 amino acids from neurofibromin located outside the conserved domain contains catalytic activity. In our hands this protein is unstable and has no catalytic activity. Thus, we believe that we have defined the true minimal domains of p120(GAP) (GAP-273, residues Met714-His986) and neurofibromin (NF1-230, residues Asp1248-Phe1477), which can be expressed via LMM fusion vectors in Escherichia coli and isolated in high purity.
Ahmadian
M R
MR
Max-Planck-Institut für Molekulare Physiologie, Abteilung Strukturelle Biologie, Rheinlanddamm 201, 44139 Dortmund, Germany.
Wiesmüller
L
L
Lautwein
A
A
Bischoff
F R
FR
Wittinghofer
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D016384:Consensus Sequence; D017931:DNA Primers; D004926:Escherichia coli; D020690:GTPase-Activating Proteins; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D025542:Neurofibromin 1; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020703:ras GTPase-Activating Proteins
D017931:DNA Primers; D020690:GTPase-Activating Proteins; D025542:Neurofibromin 1; D011506:Proteins; D011993:Recombinant Fusion Proteins; D020703:ras GTPase-Activating Proteins; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)