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Biochemical role of carboxylic acids in the L5 loop of human Eg5 kinesin
Author(s) -
Kim Sunyoung,
Sebring Sarah,
Saunders T. Randall,
Wojcik Edward J.,
Walker Richard
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a465-b
Subject(s) - allosteric regulation , kinesin , atp hydrolysis , chemistry , biophysics , nucleotide , stereochemistry , motor protein , amino acid , biochemistry , microtubule , enzyme , microbiology and biotechnology , biology , atpase , gene
Monastrol induces collapse of the mitotic spindle via allosteric inhibition of human Eg5, a Kinesin‐5 motor protein required for cell division. Solely in the presence of ATP and monastrol, acidic residues in Eg5 incur chemical and/or structural changes as revealed by FT‐IR spectroscopy. Glu116 and Glu118 in the L5 loop of Eg5 were targeted for further examination, as these amino acids are localized in the allosteric and nucleotide‐binding pockets, respectively: residues spatially close to one another may elicit synergistic functional effects arising from sidechain orientation, chemistry, or accessibility. Conservative substitution of both glutamates to aspartates, permitting similar chemistry yet shortening sidechain length, results in an Eg5 motor domain with lower ATP hydrolysis activity in the presence of microtubules. Further, this double‐mutant Eg5 yields alterations in difference FT‐IR spectra of Eg5:monastrol:ATP samples, defining vibrational modes directly attributable to Glu116/Glu118. This study demonstrates that Glu116 and/or Glu118 undergo structural changes selectively in the presence of ATP and monastrol. Modification of sidechain length of these two residues impacts not only ATP hydrolysis, but also the dynamic conformational changes that occur upon allosteric inhibition. We underscore that this direct experimental evidence supports the hypothesis that the L5 loop is involved both in ATP hydrolysis and in monastrol inhibition. Supported by ASPIRES (SK, EW & RW), NIH (GM066328 to EW) and NSF (MCB‐0130910 to RW).