Structure, function and inhibition of the two- and three-domain 4Fe-4S IspG proteins

Abstract

IspG is a 4Fe4S protein involved in isoprenoid biosynthesis. Most bacterial IspGs contain two domains: a TIM barrel (A) and a 4Fe4S domain (B), but in plants and malaria parasites, there is a large insert domain (A*) whose structure and function are unknown. We show that bacterial IspGs function in solution as (AB) 2 dimers and that mutations in either both A or both B domains block activity. Chimeras harboring an A-mutation in one chain and a B-mutation in the other have 50% of the activity seen in wild-type protein, because there is still one catalytically active AB domain. However, a plant IspG functions as an AA*B monomer. We propose, using computational modeling and electron microscopy, that the A* insert domain has a TIM barrel structure that interacts with the A domain. This structural arrangement enables the A and B domains to interact in a “cup and ball” manner during catalysis, just as in the bacterial systems. EPR/HYSCORE spectra of reaction intermediate, product, and inhibitor ligands bound to both two and three domain proteins are identical, indicating the same local electronic structure, and computational docking indicates these ligands bridge both A and B domains. Overall, the results are of broad general interest because they indicate the insert domain in three-domain IspGs is a second TIM barrel that plays a structural role and that the pattern of inhibition of both two and three domain proteins are the same, results that can be expected to be of use in drug design.

Atomic-Resolution Structures of Discrete Stages on the Reaction Coordinate of the [Fe 4 S 4 ] Enzyme IspG (GcpE)

“…In response, a variety of diphosphate-containing inhibitors have been designed and screened against the enzyme [4, 13, 17-19]. Several of them were shown to exhibit activity against both IspG as well as IspH, the downstream catalyst and final component of the MEP pathway [18, 20].…”

“…With the exception of [Fe 4 S 4 ] cluster coordination, the respective active site cavities also differ accordingly. For both enzymes, however, compounds with an increased carbon chain length and/or sterically more demanding substituents have been found to be less potent inhibitors [17] and could not be visualized as complex structures in our X-ray crystallographic studies.…”

Integrated Approach to Structure-Based Enzymatic Drug Design: Molecular Modeling, Spectroscopy, and Experimental Bioactivity

“…506 used an integrated approach to demonstrate that bacterial IspGs function in solution as (AB) (2) dimers and that mutations in either both A or both B domains block activity. Chimeras harboring an A mutation in one chain and a B mutation in the other have 50% of the activity seen in the wild-type protein as there is still one catalytically active AB domain.…”

Inhibition of the 4Fe–4S proteins IspG and IspH: an EPR, ENDOR and HYSCORE investigation

“…The A* domain has essentially no sequence conservation between different organisms, but has been proposed to again adopt a TIM (triosephosphate isomerase) barrel fold and act in a primarily structural capacity, enabling the A and B domains in a monomer to come together and function catalytically. 9 IspH is also modular and contains three αβ domains, the 4Fe-4S cluster being buried at the center of the trimeric structure. 4,5 …”