Structure-Guided Functional Characterization of Enediyne Self-Sacrifice Resistance Proteins, CalU16 and CalU19

• Published in: ACS chemical biology Vol. 9; no. 10; pp. 2347 - 2358
• Main Authors: Elshahawi, Sherif I, Ramelot, Theresa A, Seetharaman, Jayaraman, Chen, Jing, Singh, Shanteri, Yang, Yunhuang, Pederson, Kari, Kharel, Madan K, Xiao, Rong, Lew, Scott, Yennamalli, Ragothaman M, Miller, Mitchell D, Wang, Fengbin, Tong, Liang, Montelione, Gaetano T, Kennedy, Michael A, Bingman, Craig A, Zhu, Haining, Phillips, George N, Thorson, Jon S
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 17.10.2014
• Subjects: Anti-Bacterial Agents - pharmacology; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Crystallography, X-Ray; Drug Resistance, Bacterial - genetics; Enediynes - pharmacology; Genomics - methods; Lipids - chemistry; Micromonospora - growth & development; Micromonospora - metabolism; Models, Molecular; Molecular Structure; Multigene Family; Mutagenesis, Site-Directed; Mutation - genetics; Protein Structure, Tertiary; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
• ISSN: 1554-8929; 1554-8937
• DOI: 10.1021/cb500327m

Calicheamicin γ1 I (1) is an enediyne antitumor compound produced by Micromonospora echinospora spp. calichensis, and its biosynthetic gene cluster has been previously reported. Despite extensive analysis and biochemical study, several genes in the biosynthetic gene cluster of 1 remain functionally unassigned. Using a structural genomics approach and biochemical characterization, two proteins encoded by genes from the 1 biosynthetic gene cluster assigned as “unknowns”, CalU16 and CalU19, were characterized. Structure analysis revealed that they possess the STeroidogenic Acute Regulatory protein related lipid Transfer (START) domain known mainly to bind and transport lipids and previously identified as the structural signature of the enediyne self-resistance protein CalC. Subsequent study revealed calU16 and calU19 to confer resistance to 1, and reminiscent of the prototype CalC, both CalU16 and CalU19 were cleaved by 1 in vitro. Through site-directed mutagenesis and mass spectrometry, we identified the site of cleavage in each protein and characterized their function in conferring resistance against 1. This report emphasizes the importance of structural genomics as a powerful tool for the functional annotation of unknown proteins.