ROBERT H. KRETSINGER

Contact Information

Research Interests

We have determined the crystal structures of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS) and of its near homolog, 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDOPS), in various liganded states. Comparisons and evaluations of these structures have revealed the coordination of PEP, the twisting from planarity of the PEP by the enzymes as an apparent initial step in the reaction of PEP with E4P (in DAHPS) or with A5P (in KDOPS). The coordinations of E4P and of A5P have also been determined as have their initial interactions with PEP in the first step of condensation. The role of Mn²⁺ in coordinating PEP in DAHPS is explained, as is the lack of metal binding in KDOPS with compensatory coordination of PEP by Lys and Arg.

We have determined the crystal structure of DAHPS from E. coli complexed with its cognate feedback inhibitor, Phe, and defined the specificity and mechanism of feedback inhibition. The absence of feedback inhibition in KDOPS is rationalized. We have determined the structure of DAHPS from Thermatoga maritima. It contains an extra domain. DAHPS^Tma is subject to inhibition by Phe; however, the loop comprising the Phe binding site in DAHPS^Eco is not present.  DAHPS^Tma does contain an extra domain that resembles flavodoxin; upon binding Phe this domain moves ~15; this indicates a different mode of feedback inhibition. Evaluation of the amino acid sequences of different DAHPSs suggests at least four distinct modes of allosteric coupling.

In collaboration with Julie Sando (Department of Anesthesiology) we have grown and characterized several two dimensional crystals of both protein kinases Cd and ß as grown on phospholipid monolayers. We have extended our electron micrographic studies to compute three dimensional reconstructions of the regulatory domain of PKCd and of intact PKCd. We will model its four domains whose individual crystal structures are known. We are also evaluating the changes in conformation of PCK's that accompany the binding of substrate analogs and of effectors (as diacylglycerol and calcium) of this key enzyme in cell signaling.

In collaboration with Sven Hovmöller (Physical Chemistry, Stockholm,Sweden) and Roger Ison (Mantic Software - Loveland, CO) we have initiated a knowledge based program to predict ab initio the tertiary structures of proteins from their amino acid sequences.

In collaboration with Carl Creutz (Pharmacology) we determined the crystal structure of the calcium bound form of annexin VI to 2.9 Å resolution. The structure consists of two discs (domains 1-4 and 5-8) that are tipped 90° to one another. Under some conditions the two discs lie on a lipid monolayer both in the same orientation; under other conditions disc 5-8 is flipped over to antiparallel, as seen in 3D reconstructions from electron micrographs of 2D crystals. We have also determined the crystal structure of the T356D mutant that mimics phosphorylation at Thr356 and have correlated the subtle changes in structure with changes in physical properties of annexin VI.

In 1974 I presented a general hypothesis that has guided much of our subsequent research:

1. Resting cells maintain the concentration of free Ca²⁺ ions in the cytosol between 10^-8 and 10^-7 M.
2. The sole function of calcium in the cytosol is to transmit information.
3. The target of calcium, functioning as a second messenger, is a protein(s) in the cytosol.
4. Calcium modulated proteins are homologs, containing EF-hands.
5. Cells extrude calcium so they can use phosphate as their energy currency; Ca₃(PO₄)₂ is insoluble.

We have completed an extensive analysis of the evolution of 1000 EF-hand homolog proteins and identified 78 distinct subfamilies. Some subfamilies, such as calmodulin, troponin C, essential light chains, and regulatory light chains of myosin, are congruent; all evolved from a single protein that contained four EF-hand domains. Others, like calpain, evolved from a single EF-hand domain by recent gene duplications and fusions. Most other subfamilies have complex evolutionary histories involving multiple gene translocations and splicings. Intron sites are found at many positions within domains and appear not to have facilitated the multiple domain swappings involved in the evolution of this complex subfamily.

Three dimensional reconstruction of protein kinase C (left) and of its regulatory domain (right) from electron micrographs of two dimensional crystals. The inferred catalytic domain (green) faces the cytosol; the C1 and C2 domains face to cell membrane. (Solodukin, Kretsinger, and Sando, J. Biol. Chem. 2006, submitted)

Scientific Publications since 2003

1. Minks, C., Kretsinger, R.H. and Creutz, C.E. "Crystal Structure and Functional Characteristics of the T356D Mutant of Annexin VI" Biochemistry (2003) 42 620-630.

2. Shumilin, I.A. Bauerle, R., and Kretsinger, R.H. " High resolution structure of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase reveals a twist in the plane of bound phosphoenolpyruvate" Biochemistry (2003) 42 3766-3776.

3. Nakayama, S., Kawasaki, H., and Kretsinger, R.H. "Parvalbumin" in A. Messerschmidt, M. Cygler and W. Bode (eds.) Handbook of Metalloproteins, vol 3 (John Wiley & Sons, Ltd.) (2004) 42 501-508.

4. Kretsinger, R.H., Hovmöller, S., and Ison, R.E. "Prediction of Protein Structure" Meth. Enz. (2004) 383 1-27.

5. Shumilin, I.A., Bauerle, R., Wu, J., Woodard, R.W., and Robert H. Kretsinger "Crystal Structure of the Reaction Complex of 3-deoxy-D-arabino-heptulosonate-7-phosphate Synthase from Thermotoga maritima Refines the Catalytic Mechanism and Indicates a New Mechanism of Allosteric Regulation" J. Mol. Biol. (2004) 341 455-466.

6. Kretsinger, R.H., "Proteins and the Flow of Information in Cellular Function" in Molecular Imaging: FRET Microscopy and Spectroscopy pp1-15, eds. A. Periasamy and R.N. Day, Oxford University Press (2005).

7. Ison, R.I., Hovmöller, S. and Kretsinger, R.H. "Proteins and their Shape Strings" IEEE Eng. Med. Biol. (2004) 24 41-49.

8. Matsushima, N., Kuroki, Y., Enkhbayar, P., Kamiya, M. and Kretsinger, R.H. "Leucine-rich Repeats (LRRs) Proteins Associated with Human Diseases Such as Crohn's Disease, Bernard-Soulier Syndrome (BSS), CINCA Syndrome and Schizophrenia"
Drug Design Reviews. (2005) 2 305-322.

9. Matsushima, N., Tachi, N., Kuroki, Y., Enkhbayar, P., Osaki, M., Kamiya, M. and Kretsinger, R.H. "Structural analysis of leucine rich repeat (LRR) variants in proteins associated with human diseases" Cell. Mol. Life Sci. (2005) 62 2771-2791.

Book Reviews

Kretsinger, R.H. review "People and Place, Information and Structure" of " Designs for Life: Molecular Biology after World War II" by Soraya de Chadarevian in Cell (2003)
113 5-6.

updated 2-14-08