Douglas H. Ohlendorf, Fellow

Structural Biology of Macromolecules

The work in the Ohlendorf laboratory involves creating and refining structural models of macromolecules using diffraction data, geometric constraints, and/or structures of homologous molecules. The goal of this research is to produce models that are sufficiently accurate to gain insight into the structural foundations for their biological functions. The predictions made by these models were checked through biophysical analysis of complexes and mutants of targeted proteins.

In recent work, the structural analysis of macromolecules was carried out in order to understand the structural basis of how macromolecules function. The current focus is on seven proteins—protocatechuate 3,4-dioxygenase (PCD), homoprotocatechuate 2,3-dioxygenase (HPCD), 1,2-catechol dioxygenase (CTD), toxic shock syndrome toxin-1 (TSST- 1), streptococcal pyrogenic exotoxin A (SPEA), exfoliative toxin A (ETA), and exfoliative toxin B (ETB). The researchers used the computational resources of the Supercomputing Institute to refine structures of substrate and inhibitor complexes of PCD; to solve and refine the structures of HPCD, CTD, SPEA, ETA, ETB, and mutants to TSST-1, ETA, ETB, and PDC; and to model the structures of other dioxygenases and superantigens.

Work on the PCD system involves PCDs from three organisms—Pseudomonas putida, Brevibacterium fuscum, and Acinetobacter calcoaceticus. Dioxygenases carry out a unique reaction in which molecular oxygen is induced to cleave an aromatic ring. This reaction is important because it is used by bacteria to degrade a number of toxic compounds that contaminate the environment. The reaction is interesting because cleavage of a stable aromatic ring occurs without the use of exotic cofactors. In PCD the cleavage is between the vicinal hydroxyls (intradiol) and is accompanied by a non-heme Fe⁺³ ion.

As with PCD, in HPCD a single metal ion is used to cleave an aromatic ring. Unlike PCD, this cleavage is adjacent to the vicinal hydroxyls extradiol) and utilizes a non-heme Fe⁺² ion or a Mn⁺² ion. Work on this protein is focused on metal specificity as well as substrate selectivity.

Like PCD, CTD is an intradiol dioxygenase incorporating both atoms of molecular oxygen into an organic substrate using a non-heme ferric ion. However, where the PCDs being studied contain six or more protomers each containing two polypeptide chains, CTD is a dimer of a single poly peptide chain. In addition, the substrate specificity is different in that CTD does not tolerate a carboxylate group on the aromatic ring para and meta to the hydroxyls. It is possible to select for mutants of CTD that have extradiol activity.

Toxic shock syndrome toxin-1 (TSST-1), from Staphylococcus aureus, is the causative agent in toxic shock syndrome and is a member of a family of pyrogenic toxins that have been referred to as superantigens. Typically 0.0001%– 0.01% of helper CD4⁺ T-cells respond when presented with an antigen. However, when a superantigen such as TSST-1 is present, as many as 20% of these helper Tcells respond by releasing large amounts of cytokines producing fever, vomiting, diarrhea, shock, and sometimes death. Through the production of mutants of TSST-1, the researchers hope to be able to modulate these activities.

SPEA from Streptococcus pyogenes is the causative agent of toxic shock-like syndrome. This syndrome is 60–80% fatal and is the disease that killed the entertainer Jim Henson. Through analysis of the structure of SPEA in light of those of other superantigens (e.g., TSST-1), the researchers hope to discover the structural foundations for biological activities.

Effoliative toxins A (ETA) and B (ETB) from S. aureus are the causative agents in staphylococcal scalded skin syndrome. Although they are superantigens, there is no structural homology with other superantigens. In addition, their exfoliative activities depend upon a putative proteolytic activity.

Research Group and Collaborator

Mohammed Badasso, Research Associate
Abdul R. Bahar, Supercomputing Institute Undergraduate Intern
C. Kent Brown, Graduate Student Researcher
Cathleen A. Earhart, Research Associate
Heidi Flashinski, Undergraduate Student Researcher
Deborah Garrido, Undergraduate Student Researcher
Kyeong Soo Jeong, Graduate Student Researcher
Nick Kaster, Staff John McCormick, Faculty Collaborator
Alexa Pragman, Graduate Student Researcher
Greg Vath, Graduate Student Researcher
Matthew Vetting, Graduate Student Researcher