This project is focussed on the elucidation of structures of proteins in the secretory pathway. The key protein in vesicle formation is the ADP ribosylation factor that sequesters the proteins required for vesicle formation to the membrane. The protein behaves like an on/off switch where the GDP form is soluble in the cytosol and the GTP form is membrane associated. One of the elements that is essential for membrane association is a myristyl group at the N-terminus of the protein, without which membrane association does not occur. So far, the structure of the human ARF1 has been determined, and structures of yeast ARF1 and ARF2 are underway. In addition to the ARF's, cells also contain ARF-related proteins, called ARL's, that seem to have a function similar to that of ARF, but not identical. We have crystallized ARL1 from drosophila in hopes of obtaining a structure that might indicate what the differences between ARL and ARF are, and how they relate to cellular function. These crystals are too small for in-house data collection. During the trip to CHESS (8.-11. April 1998) a number of ARL crystals were screened, however, none of them diffracted to high enough resolution (better than 8A) for useful data collection. The enzyme responsible for myristylation at the N-terminus of the ARF's and ARL's is N-myristyl transferase. The structure of this enzyme from yeast has been determined, but is sufficiently different from the human enzyme by sequence comparison that we are determining the structure of the human enzyme. The protein crystallizes slowly and produces very small crystals that do not diffract well enough for in-house data collection. A 85% complete data set (to 3A resolution) of the hNMT was obtained at CHESS (8.-11. April 1998) under cryo conditions. We are in the process of using the yeast NMT coordinates to these data in molecular replacement attempts. So far, we have obtained two promising solutions that have to be analyzed further before we have a definitive answer.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Biotechnology Resource Grants (P41)
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Cornell University
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Kozlov, Guennadi; Wong, Kathy; Gehring, Kalle (2018) Crystal structure of the Legionella effector Lem22. Proteins 86:263-267
Ménade, Marie; Kozlov, Guennadi; Trempe, Jean-François et al. (2018) Structures of ubiquitin-like (Ubl) and Hsp90-like domains of sacsin provide insight into pathological mutations. J Biol Chem 293:12832-12842
Xu, Jie; Kozlov, Guennadi; McPherson, Peter S et al. (2018) A PH-like domain of the Rab12 guanine nucleotide exchange factor DENND3 binds actin and is required for autophagy. J Biol Chem 293:4566-4574
Dean, Dexter N; Rana, Pratip; Campbell, Ryan P et al. (2018) Propagation of an A? Dodecamer Strain Involves a Three-Step Mechanism and a Key Intermediate. Biophys J 114:539-549
Chen, Yu Seby; Kozlov, Guennadi; Fakih, Rayan et al. (2018) The cyclic nucleotide-binding homology domain of the integral membrane protein CNNM mediates dimerization and is required for Mg2+ efflux activity. J Biol Chem 293:19998-20007
Xu, Caishuang; Kozlov, Guennadi; Wong, Kathy et al. (2016) Crystal Structure of the Salmonella Typhimurium Effector GtgE. PLoS One 11:e0166643
Cogliati, Massimo; Zani, Alberto; Rickerts, Volker et al. (2016) Multilocus sequence typing analysis reveals that Cryptococcus neoformans var. neoformans is a recombinant population. Fungal Genet Biol 87:22-9
Oot, Rebecca A; Kane, Patricia M; Berry, Edward A et al. (2016) Crystal structure of yeast V1-ATPase in the autoinhibited state. EMBO J 35:1694-706
Lucido, Michael J; Orlando, Benjamin J; Vecchio, Alex J et al. (2016) Crystal Structure of Aspirin-Acetylated Human Cyclooxygenase-2: Insight into the Formation of Products with Reversed Stereochemistry. Biochemistry 55:1226-38
Bauman, Joseph D; Harrison, Jerry Joe E K; Arnold, Eddy (2016) Rapid experimental SAD phasing and hot-spot identification with halogenated fragments. IUCrJ 3:51-60

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