Grant Lab Research

The regulation of biological processes is critical to sustaining life. The control mechanisms that moderate biological processes may be thought of as being just as important as the processes themselves because life could not be sustained if this metabolic framework was out of control.

In any organism, regulatory processes are widely distributed and exceedingly complex. Thus, it is still virtually impossible to study regulatory systems at the molecular level in a general sense. Proteomics and Systems Biology approaches are starting to give us information on which components interact within a cell or organism. However, understanding the molecular basis of that interaction will rely on understanding the details of each component. In a general sense, understanding of all proteins, from those that appear relatively simple to the very complex, will benefit from what is learned from research that explores the fundamental mechanisms of the interplay of elements that contribute to specific protein function.Only in understanding the complexity and interplay of structural elements in specific, molecularly well-defined systems can we hope to build our knowledge to the level of understanding how they can be utilized to prevent disease and for the betterment of the human condition.

We are pursuing our goals by focusing on proteins whose functions are regulated by the ACT domain and related domains. Some of the tools that we are utilizing to advance our studies include Site-directed Mutagenesis, Fluorescence Spectroscopy, and Steady-State and Transient Kinetic analysis. We also utilize X-ray crystallography analysis through collaborations.

The ACT domain

Our laboratory initiated research that led to the determination of the structure of the prototypical ACT domain in E. coli D-3-phosphoglycerate dehydrogenase in 1995.

The ACT domain is a babbab motif that binds small molecules for the regulation of activity and is found in a growing number of proteins. The wide-spread existence of the ACT domain was originally described by Aravind and Koonin (J. Mol. Biol. 287, 1023-1040, 1999.) as the result of an iterative sequence database search with the small subunit of acetolactase synthetase (IlvN). This search found sequence similarities with a substantial number of proteins. They named this domain the "ACT" domain after the first letters of three of these proteins, Asparokinase, Chorismate mutase, and TyrA.

In addition to being found in a wide variety of proteins, ACT domain are also found associated in many different configurations in these proteins. Some examples are provided below.

ACT Domain Structure and Configuration. The structure of the ACT domains from four proteins are presented for comparison. The proteins are PGDH, D-3-phosphoglycerate dehydrogense from E. coli; Nik R, Nickel responsive transcription factor NikR from E. coli; ATP-PRT, ATP phosphribosyltransferase from M. tuberculosis; YkoF, YkoF gene product of thiamine binding protein from B. subtilus. The babbab motif is shown in the top row along with the bound ligands. The bound ligands are represented as ball and stick models except for nickel which is shown as a sphere. The bottom row shows the different quaternary structure configurations of the different proteins. PGDH is a tetramer where two sets of two ACT domains form interfaces at opposite ends of the protein. NikR is a tetramer where all four ACT domains interface as shown. ATP-PRT is a hexamer where two sets of three ACT domains form interfaces at opposite ends of the protein. YkoF is a dimer where each subunit contains two ACT domains oriented as shown.

The Phosphoglycerate Dehydrogenase Family

Our research focuses on the question of how ACT domains regulate protein function. We are presently pursuing this by studying the regulatory mechanisms of the Phosphoglycerate dehydrogenase (PGDH) family. PGDH catalyzes the first committed step in serine biosynthesis by converting the glycolytic intermediate D-3-phosphoglycerate to hydroxypyruvic acid phosphate utilizing NAD+ as a cofactor. In E. coli and M. tuberculsosis, the enzyme is inhibited by L-serine, the end product of the pathway, in a non-competitive cooperative manner.

Clinically, D-3-phosphoglycerate dehydrogenase deficiency is responsible for severe encephalopathy of prenatal onset and very low serine levels in the cerebrospinal fluid.

PGDH exists in at least three different basic structural motifs that do not appear to be strictly specific for organism type (see below). The PGDH of some bacteria and some lower eucaryotes, such as yeast, Leishmania, and Neurospora are structurally similar to the E. coli enzyme. In addition to well defined substrate and nucleotide binding domains, they possess a homologous C-terminal domain that is involved in effector binding (L-serine) and regulation of activity. This is the "ACT domain." Other bacteria and higher order eucaryotes, including mammals, possess a large polypeptide insertion in their C-terminal segment immediately following the substrate-binding domain. We call this the "intervening domain." In M. tuberculosis PGDH the intervening domain possesses a distinct anion binding site.

A third motif, which lacks the C-terminal regulatory domain altogether, is also found in some bacteria, including some, such as M. tuberculosis (Ser A2 PGDH), that also produce PGDH with the extended C-terminal motif. Recently, a variation of this third motif has been recognized in the parasite Entamoeba histolytica that appears to utilize a lysyl residue rather than a histidyl residue at the active site for proton transfer.

PGDH Structural motifs. The polypeptide arrangement of PGDH falls into four general categories composed of 3 structural motifs. Top: PGDH from some bacteria and simple eucaryotes consist of three distinct domains called the cofactor or nucleotide binding domain which also contains the active site histidine (H), the substrate binding domain and the serine binding regulatory or ACT domain. The substrate binding domain is a distinct structural entity formed by two polypeptide segments which flank the nucleotide binding domain in the sequence. These proteins form a second domain interface at the regulatory domain and exist as tetramers. They also contain an invariant tryptophan (Trp 139) that inserts into a pocket on the adjacent subunit at the nucleotide binding domain interface. TopMiddle: PGDH from other bacteria and higher order procaryotes such as mammals contain a large insert (intervening domain) between their substrate binding domain segment and the segment displaying homology with the regulatory domain. They also contain the invariant tryptophan (W). Bottom Middle: Some organisms also contain a PGDH that is devoid of a regulatory domain segment. They exist as dimers and may or may not retain the Trp. These organisms appear to always contain PGDH with one of the other two motifs. Bottom: A few organisms have been found to contain a PGDH where the active site histidine (H) has been replaced by a lysine residue (K). They are also dimers and are missing the Trp residue. The symbol "~" indicates that the N-termini are of various lengths.

	E. coli PGDH is a tetramer of identical subunits where each subunit is composed of three well-defined domains referred to as the substrate binding, nucleotide binding, and regulatory (ACT) domains. L-Serine binds at the interface between regulatory domains. Two regulatory domain interfaces exist in the tetramer and each interface binds two serine molecules. We have shown that the first two serines bind at opposite interfaces and that the binding of these two serines is sufficient to produce the cooperative allosteric effect on the inhibition of enzyme activity. Two additional serines may bind, but do not contribute to the inhibition. In addition, production of asymmetric hybrid tetramers of PGDH have shown that it displays half-of-the-sites activity and that the binding of a single serine completely inhibits the operative active site of the dimer to which it binds as well as producing an approximately 33% inhibition of the operative active site on the adjacent dimer to which it has no binding interaction. This is the basis for the positively cooperative regulation of enzyme activity.

	M. tuberculosis PGDH contains analogous substrate and nucleotide binding domains, a well defined regulatory or ACT domain, and a new domain adjacent to the regulatory domain that we term the "intervening" domain. We have elucidated what appears to be a new ligand binding site that potentially links the regulatory (ACT) domain to the intervening domain. This very positively charged pocket is well defined structurally and clearly appears to be poised for the binding of a negatively charged metabolite

The crystal structure of M. tuberculosis PGDH has also revealed a very surprising and seemingly unprecedented level of asymmetry in the tetramer. Two subunits are present with a completely different orientation of domains as compared to the other two subunits. This is manifested by an approximately 180 º inversion of the intervening domain around a hinge between it and the substrate binding domain. The result is that the intervening domain can form two different interfaces with the other subunits, depending on which orientation it takes.

M. tuberculosis PGDH as a potential drug target

Approximately 3 million annual deaths from tuberculosis were reported in the 1990s. It has been estimated that as much as one-third of the world's population harbor the tuberculosis pathogen and multi-drug resistant tuberculosis is on the increase. Tuberculosis will be a leading cause of mortality worldwide in the 21st century.

The observations that M. tuberculosis PGDH is an essential gene product and that its activity is inhibited by L-serine while human PGDH is not, makes it a potential candidate for drug-targeting. In addition, we have discovered an additional small molecule binding site in M. tuberculosis PGDH that may also be a potential target.

We are pursuing this aspect using computer assisted virtual screening approaches for small molecule binding and we plan to screen actual small molecule libraries in the future. Potential inhibitory molecules will then be tested in the laboratory.

This page was last updated Wednesday, June 6, 2007