Colibactin is a genotoxin produced by gut microbes that has been implicated in the development of diseases such as colitis and colorectal cancer. The biosynthesis of this natural product follows a prodrug resistance mechanism, in which bacteria assemble a non-toxic precursor (pre-colibactin) in the cytoplasm and activation to the toxic species occurs only after pre-colibactin has been exported to the periplasm. This activation is mediated by ClbP, a serine hydrolase that cleaves off part of pre-colibactin and, as a result, predisposes it to undergo a critical cyclization event. ClbP is an inner membrane-bound enzyme with an N-terminal periplasmic domain containing the catalytic residues followed by a C-terminal domain of three transmembrane (TM) helices. Although the structure of the isolated periplasmic domain has been elucidated, it cannot be used to derive any significant mechanistic insights because ClbP mutants lacking one or all transmembrane helices are functionally inactive. Furthermore, only one TM helix is sufficient for successful localization of the protein to the inner membrane, suggesting that the role of the transmembrane domain is not limited to membrane anchorage. My proposed research plan aims to further our understanding of pre-colibactin binding and turnover by ClbP through structural and biochemical studies of the full-length enzyme.
Aim 1 involves the use of lipid mesophase-based crystallization techniques to obtain high-resolution structures of the enzyme in absence or presence of substrate analogs. Preliminary results from this aim suggest that the active site reported in the literature may be incomplete, and that residues in the linker between TM helices 2 and 3 (TM2-TM3) may complete the pre-colibactin binding site in the full-length enzyme. Based on these observations, Aim 2 addresses the functional relevance of residues in the TM2-TM3 linker as well as of residues in the interface between the periplasmic and the C-terminal TM domain. Finally, Aim 3 investigates whether a dimeric interface observed in the crystal contacts of our preliminary model is physiologically relevant, and whether it is important for activity. The mechanistic insights gained from this proposal will allow us to better understand colibactin maturation and will likely be relevant to understanding less studied prodrug-cleaving peptidases. Also, the full-length structure proposed here will enable the rational design of inhibitors of ClbP, which could be useful pharmacological agents or tool compounds for studying the dynamics of gut microbiome populations. The collaboration between the labs of Dr. Rachelle Gaudet and Dr. Emily Balskus is the ideal setting to conduct the research at the interface between structural and chemical biology detailed in this proposal. Training in the techniques of membrane protein structure, biochemistry and enzymology, as well as the professional development these faculty mentors provide will allow me to become a capable independent scientist.

Public Health Relevance

Colibactin is a genotoxin produced by gut microbes that has been implicated in the development of diseases such as colitis and colorectal cancer. The ClbP peptidase performs an essential step in the biosynthesis of this metabolite, in which a non-toxic precursor is converted into the active species, yet its mechanism is not completely understood. The structural and biochemical studies proposed here will provide mechanistic insights that will be instrumental to understand a novel family of bacterial peptidases as well as to rationally design inhibitors of pharmacological interest.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
1F31GM133097-01
Application #
9759048
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Brown, Patrick
Project Start
2019-09-15
Project End
Budget Start
2019-09-15
Budget End
Support Year
1
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Harvard University
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138