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Research

Summary

Our goals are to understand the structural properties of biological molecules and to relate these structures to their biological functions. Toward these goals, we study the structures of protein-nucleic acid complexes in order help to explain the principles that underlie interactions between nucleic acids and proteins, as well as to increase our understanding of the role of water in mediating intermolecular interactions.

We have also established a program in structural bioinformatics. A key focus of this work has been to establish methods to collect, archive, and analyze structural data. In 1991, we created the Nucleic Acid Database (NDB). In October 1998, the management of the Protein Data Bank (PDB) was moved to the Research Collaboratory for Structural Bioinformatics, of which Rutgers is the lead site.

Bioinformatics: The PDB and the NDB

We are responsible for the continued development and operation of two archival databases: the Protein Data Bank (PDB; http://www.pdb.org) and the Nucleic Acid Database (NDB; http://ndbserver.rutgers.edu)

The PDB is managed by two members of the Research Collaboratory for Structural Bioinformatics (RCSB): Rutgers University and the San Diego Supercomputer Center and the Skaggs School of Pharmacy and Pharmaceutical Sciences at the University of California, San Diego. It is a member of the wwPDB (www.wwpdb.org).

The PDB is the single worldwide depository of information about the three-dimensional structures of large biological molecules, including proteins and nucleic acids. These are the molecules of life that are found in all organisms including bacteria, yeast, plants, flies, and mice, and in healthy as well as diseased humans. Understanding the shape of a molecule helps to understand how it works.

The Nucleic Acid Database assembles and distributes structural information about nucleic acid structures. The core of the NDB is the relational database of the structural data of three-dimensional nucleic acid-containing crystal structures.

Binary and Ternary Complexes with Catabolite Activating Protein (CAP)

Although CAP makes no direct contact with the consensus base pair T:A at position 6 of DNA half site 5’-A₁A₂A₃T₄G₅T₆G₇A₈T₉C₁₀T₁₁-3’, it exhibits strong specificity for T:A at position 6. We have determined and compared crystallographic structures of CAP in complex with DNA sites having T:A at position 6 or the non-consensus base pair C:G at position 6, analyzing two DNA fragment lengths and two crystal forms. The results indicate that indirect readout in this system is mediated through altered DNA deformability.

Substitution of the carboxylate side chain of Glu181 by the one-methylene-group-shorter carboxylate side chain of Asp results in a new DNA binding specificity at position 6 of the DNA half site. Thus, whereas wild-type CAP prefers T:A at position 6, [181Asp]CAP prefers C:G at position 6. This specificity is observed in vitro and in vivo and is large in magnitude. To define the basis for this unexpected change in specificity, we have carried out structural studies of [181Asp]CAP in complex with DNA. The results indicate that the Glu Asp substitution eliminates the primary kink and thus eliminates indirect-readout-based specificity for T:A at position 6.
CAP activates transcription at P_lac and other promoters through interactions with the RNA polymerase alpha-CTD subunit. We have determined the structure of CAP in complex with DNA and alpha-CTD at 3.3Å. This structure provides the first high-resolution view of a transcriptional activator in complex with a functional target within the general transcription machinery.

DNA binding proteins
In collaboration with Drs. Jannette Carey (Princeton University) and Cathy Lawson, we have determined the structure of E. coli trp repressor (trpR) in crystals grown from 30% aqueous isopropanol. Our current structural view of trpR as a compact alpha-helical homodimer is based largely on experiments performed in high salt. In the new structure, individual trpR subunits have a dramatically extended conformation and undergo domain swapping. Similar extended subunit structures may contribute to trpR aggregation phenomena.

Quantitative Rules for Discerning DNA-Binding Proteins Relative to the Site of DNA Interaction

We are working to develop simple and quantitative rules that can be used to identify proteins that are likely to bind to DNA. The method used here creates a statistical model of a protein fold relative to the site of DNA interaction. The statistical model is based on measurements that treat the secondary structure elements near the site of interaction as individual units. Analysis of the measurements for the Helix-Turn-Helix (HTH) DNA-binding motif show that they can be used to discern proteins with the motif with high accuracy. Further analysis will extend to other DNA-binding motifs and whole protein domains.

RNA Visualization
We have also begun to develop new visualization tools to display RNA structures. RNADraw automatically displays the tertiary interactions on a 2D format. The 2D picture provides detailed contact information between the base pairs and provides a fast and simple way to compare related structures. We are planning to use this tool to help classify the different folds in RNA.

Awards & Honors