BAHNSON RESEARCH (click here for publication list)
Structural Enzymology:
We are pursuing the structural determination of the mechanisms of several classes of enzymes and with a range of project goals: engineering organophosphate hydrolysis activity in peripheral membrane enzymes, structural mimics of interfacial bound membrane proteins, the calcium and integrin binding protein, proteins motions linked to catalysis, enzyme-substrate complexes of enoyl-CoA hydratase, and NADP dependent isocitrate dehydrogenase. This work entails solving de novo structures of medically significant enzymes and taking the new structures a step further in a quest to elucidate each enzyme's mechanism.

In one project, we are focusing on a group of human HDL and LDL associated enzymes that have direct links to atherosclerosis. These peripheral membrane proteins are of medical interest due to their connections with signal transduction and lipid remodeling, related to heart disease. Additionally, one of the systems currently under study is a promising catalyst for the detoxification of organophosphate neurotoxins. We use a combination of protein expression, site-directed mutagenesis, kinetics, homology modeling and x-ray crystallography to understand the relationship between structure and function, with a goal of either inhibiting detrimental activities or designing more specific beneficial catalytic activities.

We have also solved the structures of the crystallographic dimer of porcine pancreatic group-IB PLA2 with five coplanar phosphate anions bound. In the anion-assisted dimer structure one molecule of a tetrahedral mimic inhibitor and the five anions are shared between the two subunits of the dimer. The sn-2-phosphate of the inhibitor is bound in the active site of one subunit, and the alkyl chain extends into the active site slot of the second subunit across the subunit-subunit interface, as shown below. 

 
Remarkably, the plane defined by the contact surface is similar to the i-face of the enzyme, which has been proposed to make contact with the substrate-interface for the interfacial catalytic turnover.  Additionally, these structures not only offer a view of the active PLA2 complexed to an anionic interface, but also provide insight into the environment of the tetrahedral intermediate in the rate-limiting chemical step of the turnover cycle.  Taken together, our results offer an atomic-resolution structural view of the i-face interactions of the active form of PLA2 associated to an anionic interface.
Y. H. Pan,  T. M. Epstein, M. K. Jain, and B. J. Bahnson* (2001) Five coplanar anion binding sites on one face of phospholipase A2:  relationship to interface binding, Biochemistry, 40, 609-617.

T. M. Epstein, Y. H. Pan, S. P. Tutton, M. K. Jain, and B. J. Bahnson* (2001) The basis for k*cat impairment in prophospholipase A(2) from the anion-assisted dimer structure, Biochemistry, 40, 11411-22.

Y. Pan, B. Z. Yu, O. G. Berg, M. K. Jain and B. J. Bahnson* (2002) Crystal Structure of Phospholipase A2 in Complex with the Hydrolysis Products of Platelet Activating Factor: Equilibrium Binding of Fatty Acid and Lysophospholipid-Ether at the Active Site May Be Mutually Exclusive, Biochemistry, 41, 14790-14800.

We solved the crystal structure of the human Group-X PLA2 by molecular replacement and its i-face is shown above compared to a sPLA2 from human synovial fluid.

Y. H. Pan, B. Z. Yu, A. G. Singer, F. Ghomashchi, G. Lambeau, M. H. Gelb, M. K. Jain, and B. J. Bahnson* (2002) Crystal Structure of Human Group-X Secreted Phospholipase A2; Electrostatically Neutral Interfacial Binding Surface Targets Zwitterionic Membranes, Journal of Biological Chemistry, 277, 29086-29093.
 

Engineering Organophosphate Hydrolase Activity in Esterases

Organophosphate (OP) esters have applications ranging from chemical warfare nerve gas agents and pesticides, to treatments of medical conditions, such as glaucoma and parasite infections.  This class of compounds is highly toxic because it inactivates a family of serine hydrolases (esterases and lipases) that share a GXSXG consensus motif.  The essential serine, which is covalently modified, forms a catalytic triad with His and Glu/Asp.  The reactivity with OP compounds is enhanced due to the oxyanion hole that stabilizes the negative charge built up during the transition-state of the ester bond hydrolysis reaction.

Our long-term objective is to develop a conceptual framework for the understanding of phospho-transfer reactions and strategies toward controlling specificity and the design of novel phosphohydrolase activities.  Several structurally diverse enzyme systems are being examined in parallel to understand, at a mechanistic and structural level, potential routes to the inactivation of OP toxins.  In order to develop insights with each class of enzyme, one of our initial goals is to solve the three-dimensional structure of wild type forms bound with organophosphate ligands in their active sites.  This structural information will then be used to guide a molecular-modeling / site-directed mutagenesis approach to engineer OP-hydrolase activity.  Each of these modified enzymes will be studied kinetically to assess OP-hydrolase activity, as well as structurally to guide the next level of catalyst design.
 

The Calcium and Integrin Binding Protein (CIB)

CIB is a newly discovered protein that is a component to the signalling pathway of platelet aggregation.  The calcium binding function of CIB is analogous and homologous to the proteins calmodulin and calcineurin.  We are well on our way with the structure determination of this medically significant enzyme.  In collaboration with Ulhas Naik from the Biology department, we will extend our understanding by pursuing structures of functionally significant complexes of CIB with partner proteins. From a collaboration with Ulhas Naik from the Biology department we have CIB expressed as a GST-fusion protein. Ca-bound crystals diffract to 4.5 angstroms. Work is underway to improve diffractoin as well as grow crystals with the cytoplasmic tail of alpha-IIb bound.
 

Ordered Proteins Motions and Catalysis:

A related area of interest is to explore how enzymes may have evolved directed motions that are correlated to the reaction coordinate and are essential for their full catalytic power to be achieved.  This interest stems from my first postdoctoral fellowship where I studied hydrogen tunneling in the hydride transfer of alcohol dehydrogenase.  The figure shown below compares two mutants of ADH that show a hyperclosure of 0.5 angstroms of the alcohol binding domain relative to the cofactor binding domains.

This raises the question of whether the enzyme has evolved to use this type of ordered motion to, in this case, facilitate the hydride transfer of the reaction.

Here is a morphing of an open and closed form of ADH showing a hinge motion between cofactor and alcohol domains upon cofactor binding.  This movie is from the Database of macromolecular movements at Yale.

We are attempting to support this hypothesis using dynamic information from anisotropic temperature factor analysis of high resolution x-ray diffraction data. This work is an essential component of a complete understanding of enzyme mechanisms, how to create novel catalysts and how to develop selective and potent inhibitors of enzymes. The system that we are pursuing for this work is alcohol dehydrogenase from B. stearothermophilus. We have recently solved the structure of this thermophilic ADH, which is a tetramer as shown below.

 

Enzyme-substrate complex of enoyl-CoA hydratase:

Using molecular replacement, we have solved the structure of a true enzyme-substrate complex of the enzyme enoyl-CoA hydratase. I determined the chemical mechanism of this enzyme during my Ph.D. training. The structure of the enzyme complexed with p-dimethylamino cinnamoyl-CoA suggests that the atoms from a single water molecule are added across the double bond of the unsaturated thiol esters during the hydration reaction.  This structure combined with isotope effect work, as well as 13C NMR and resonance raman spectroscopy of enzyme bound substrates, in collaboration with Vernon Anderson, will give a very detailed description of catalysis.
B. J. Bahnson*, V. E. Anderson, and G. A. Petsko (2002) Structural Mechanism of Enoyl-CoA Hydratase: Three Atoms from a Single Water are Added in Either an E1cb Stepwise or Concerted Fashion, Biochemistry 41, 2621-2629.

R. L. D'Ordine, J. Pawlak, B. J. Bahnson V. E. Anderson* (2002) Polarization of Cinnamoyl-CoA Substrates Bound to Enoyl-CoA Hydratase: Correlation of 13C NMR with Quantum Mechanical Calculations and Calculation of Electronic Strain Energy, Biochemistry 41, 2630-2640.

NADP Dependent Isocitrate dehydrogenase

The crystal structure of the Mn-isocitrate binary ES complex was solved by MAD-phasing using Se-methionine expressed IDH protein. This represents the first mammalian crystal structure for IDH.

C. Ceccarelli, N. Grodsky, N. Aryaratne, R.F. Colman and B.J. Bahnson* (2002) Crystal Structure of Porcine Mitochondrial NADP+-dependent Isocitrate Dehydrogenase Complexed with Mn2+ and Isocitrate Insights into the Enzyme Mechanism, Journal of Biological Chemistry, 277, 43454-43462.