Northeastern University, Massachusetts, United States
Monica Ojeda (Northeastern University)| Kendra Marcus (University of California, Berkeley, Northeastern University)| Hyunbum Jang (Frederick National Laboratory for Cancer Research)| Avik Banerjee (University of Illinois, Chicago)| Lee Makowski (Northeastern University)| Ruth Nussinov (Frederick National Laboratory for Cancer Research, Tel Aviv University)| Vadim Gaponenko (University of Illinois, Chicago)| Carla Mattos (Northeastern University)
KRas4B (henceforth called KRas) is a small GTPase that regulates activation of a variety of signaling pathways, including the Raf/MEK/ERK pathway, leading to cell proliferation, survival, and differentiation. Activation of these signaling pathways is dependent on the nucleotide-bound state of KRas: GTP-bound KRas “turns on” signaling while GDP-bound KRas “turns off” signaling. Interestingly, KRas is the only one of the three Ras isoforms that interacts with Ca2+-bound calmodulin (CaM). However, attempts to crystallize the KRas/CaM complex have proven difficult due to the flexible nature of the hypervariable region (HVR) of KRas as well as the linker region of CaM. We present a model of the complex containing unprocessed full-length KRas in complex with CaM, guided by small angle x-ray scattering (SAXS), NMR nuclear overhouser effects and chemical shift perturbations. The low-resolution envelope of the complex shows that CaM interacts with KRas in an open conformation. The NOEs suggest that the HVR adopts a helical conformation in the region that interacts with CaM. Our low-resolution experimental data, together with published chemical shift perturbations lead to a model of the KRas/CaM complex that fits our SAXS envelop well (ꭓ=1.97). This model will be refined using molecular dynamics simulations.