Crystal Structure of a Human K-Ras G12D Mutant in Complex with GDP and the Cyclic Inhibitory Peptide KRpep-2d

Abstract

The Ras proteins play roles in cell differentiation, proliferation, and survival. Aberrant signaling through Ras-mediated pathways in tumor cells occurs as a result of several types of mutational damage, which most frequently affects the amino acids G12, G13, and Q61. Recently, KRpep-2d was identified as a K-Ras(G12D) selective inhibitory peptide against the G12D mutant of K-Ras, which is a key member of the Ras protein family and an attractive cancer therapeutic target. In this study, the crystal structure of the human K-Ras(G12D) mutant was determined in complex with GDP and KRpep-2d at 1.25 Å resolution. This structure revealed that the peptide binds near Switch II and allosterically blocks protein-protein interactions with the guanine nucleotide exchange factor. This discovery of a unique binding pocket provides valuable information that will facilitate the design of direct Ras inhibitors.

Targeting the Small GTPase Superfamily through Their Regulatory Proteins

“…KRpep‐2d inhibited cancer cell proliferation at 30 μ m , which is high compared to the in vitro IC 50 value. The crystal structure of KRas G12D in complex with GDP and KRpep‐2d was obtained, confirming that KRpep‐2d bound to a cleft near the switch II region . KRpep‐2d acts as an allosteric inhibitor, stabilizing the switch II region (distal to Sos‐binding site) in a conformation non‐conducive to nucleotide exchange.…”

“…[42] KRpep-2d inhibited cancer cell proliferation at 30 mm,which is high compared to the in vitro IC 50 value.T he crystal structure of KRas G12D in complex with GDP and KRpep-2d was obtained, confirming that KRpep-2d bound to ac left near the switch II region. [43] KRpep-2d acts as an allosteric inhibitor,s tabilizing the switch II region (distal to Sos-binding site) in aconformation non-conducive to nucleotide exchange.H owever,i ti sh ypothesized that some of the inhibitory effects seen with KRpep-2d could be due to the effect of the switch II conformational change on Ras-effector binding as well as Ras-GEF interactions.…”

Targeting der kleinen GTPasen über ihre regulatorischen Proteine

“…Die Kris-tallstruktur von KRas G12D in Komplex mit GDP und KRpep-2d wurde gewonnen und bestätigte,d ass KRpep-2d an einen Spalt nahe der Switch-II-Region gebunden war. [43] KRpep-2d agiert als allosterischer Inhibitor und stabilisiert die Switch-II-Region (distal zur Sos-Bindestelle) in einer Konformation, die dem Nukleotidaustausch nicht fçrderlich ist. Es wird jedoch vermutet, dass einige der inhibitorischen Effekte,die mit KRpep-2d beobachtet wurden, auf Switch-II-Konformationsänderungen bei der Ras-Effektorbindung sowie auf Ras-GEF-Interaktionen zurückzuführen sein kçnnten.…”

“…KRpep‐2d inhibierte die Krebszellproliferation bei einer Konzentration von 30 μ m. Dies ist im Vergleich zu dem In‐vitro‐IC 50 ein hoher Wert. Die Kristallstruktur von KRas G12D in Komplex mit GDP und KRpep‐2d wurde gewonnen und bestätigte, dass KRpep‐2d an einen Spalt nahe der Switch‐II‐Region gebunden war . KRpep‐2d agiert als allosterischer Inhibitor und stabilisiert die Switch‐II‐Region (distal zur Sos‐Bindestelle) in einer Konformation, die dem Nukleotidaustausch nicht förderlich ist.…”

From basic researches to new achievements in therapeutic strategies of KRAS-driven cancers

“…Two general mechanisms have been suggested for direct inhibition of RAS proteins, including decreasing the proportion of KRAS onc in its GTP state and disrupting the KRAS onc -effector interactions. To decrease KRAS onc -GTP levels, several approaches have been used, such as the inactivation of KRAS onc with small molecules or GTP analogs that facilitate GTP hydrolysis activity, interference with the nucleotide exchange process through disruption of the SOS-KRAS onc interaction, subversion of the native nucleotide preference of the KRAS onc to favor GDP over GTP, irreversible inhibition of the KRAS onc with its covalent modification, inactivation of KRAS onc in the GTP state, inhibition of intrinsic nucleotide exchange, and inhibition of nucleotide binding 40,67,74 . …”