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The basis for K-Ras4B binding specificity to protein farnesyltransferase revealed by 2 A resolution ternary complex
S B Long1, P J Casey, L S Beese
1Department of Biochemistry, PO Box 3711, Duke University Medical Center, Durham, 27710, USA.
Background:
The protein farnesyltransferase (FTase) catalyzes addition of the hydrophobic farnesyl isoprenoid to a cysteine residue fourth from the C terminus of several protein acceptors that are essential for cellular signal transduction such as Ras and Rho. This addition is necessary for the biological function of the modified proteins. The majority of Ras-related human cancers are associated with oncogenic variants of K-RasB, which is the highest affinity natural substrate of FTase. Inhibition of FTase causes regression of Ras-mediated tumors in animal models.
Results:
We present four ternary complexes of rat FTase co-crystallized with farnesyl diphosphate analogs and K-Ras4B peptide substrates. The Ca(1)a(2)X portion of the peptide substrate binds in an extended conformation in the hydrophobic cavity of FTase and coordinates the active site zinc ion. These complexes offer the first view of the polybasic region of the K-Ras4B peptide substrate, which confers the major enhancement of affinity of this substrate. The polybasic region forms a type I beta turn and binds along the rim of the hydrophobic cavity. Removal of the catalytically essential zinc ion results in a dramatically different peptide conformation in which the Ca(1)a(2)X motif adopts a beta turn. A manganese ion binds to the diphosphate mimic of the farnesyl diphosphate analog.
Conclusions:
These ternary complexes provide new insight into the molecular basis of peptide substrate specificity, and further define the roles of zinc and magnesium in the prenyltransferase reaction. Zinc is essential for productive Ca(1)a(2)X peptide binding, suggesting that the beta-turn conformation identified in previous nuclear magnetic resonance (NMR) studies reflects a state in which the cysteine is not coordinated to the zinc ion. The structural information presented here should facilitate structure-based design and optimization of inhibitors of Ca(1)a(2)X protein prenyltransferases.
Insights
Protein farnesyltransferase (FTase) is crucial for Ras signaling and cancer. This study reveals FTase-Ras peptide complexes, highlighting zinc
Area of Science:
- Biochemistry
- Structural Biology
- Molecular Biology
Background:
- Protein farnesyltransferase (FTase) is essential for cellular signal transduction by farnesylating proteins like Ras and Rho.
- Oncogenic K-RasB variants are implicated in most Ras-related human cancers and are high-affinity FTase substrates.
- FTase inhibition demonstrates efficacy in regressing Ras-mediated tumors in preclinical models.
Purpose of the Study:
- To elucidate the structural basis of K-Ras4B peptide substrate binding to FTase.
- To understand the role of metal ions, specifically zinc, in the prenyltransferase reaction mechanism.
- To provide insights for structure-based design of novel FTase inhibitors.
Main Methods:
- Co-crystallization of rat FTase with farnesyl diphosphate analogs and K-Ras4B peptide substrates.
- X-ray crystallography to determine the structures of four ternary complexes.
- Analysis of peptide conformation and metal ion coordination within the FTase active site.
Main Results:
- The Ca(1)a(2)X motif of K-Ras4B peptide binds in an extended conformation, coordinating active site zinc.
- The polybasic region of K-Ras4B peptide adopts a type I beta turn, enhancing substrate affinity.
- Zinc removal alters peptide conformation, while manganese binds to the diphosphate mimic.
Conclusions:
- Zinc is critical for productive Ca(1)a(2)X peptide binding, defining the active conformation.
- The observed beta-turn in NMR studies may represent a zinc-uncoordinated state.
- Structural insights facilitate the design of optimized inhibitors targeting Ca(1)a(2)X protein prenyltransferases.