Structure of Arabidopsis thaliana At1g77540 Protein, a Minimal Acetyltransferase from the COG2388 Family^,

Abstract

We describe X-ray crystal and NMR solution structures of the protein coded for by Arabidopsis thaliana gene At1g77540.1 (At1g77540). The crystal structure was determined to 1.15 Å with an R factor of 14.9% (R free = 17.0%) by multiple-wavelength anomalous diffraction using sodium bromide derivatized crystals. The ensemble of NMR conformers was determined with protein samples labeled with 15 N and 13 C+ 15 N. The X-ray structure and NMR ensemble were closely similar with r.m.s.d 1.4 Å for residues 8-93. At1g77540 was found to adopt a fold similar to that of GCN5-related N-acetyltransferases. Enzymatic activity assays established that At1g77540 possesses weak acetyltransferase activity against histones H3 and H4. Chemical shift perturbations observed in 15 N-HSQC spectra upon the addition of CoA indicated that the cofactor binds and identified its binding site. The molecular details of this interaction were further elucidated by solving the X-ray structure of the At1g77540-CoA complex. This work establishes that the domain family COG2388 represents a novel class of acetyltransferase and provides insight into possible mechanistic roles of the conserved Cys 76 and His 41 residues of this family.As part of an ongoing effort to determine the three-dimensional structures of novel eukaryotic proteins, the Center for Eukaryotic Structural Genomics (CESG 1 ) chose the gene product of † This work was supported by NIH grants P50 GM64598 and U54 GM074901 from the National Institute of General Medical Sciences (J.L.M., P.I). J.M.D. and C.E.B. acknowledge financial support from NIH grant GM059785. Use of the Advanced Photon Source and the Argonne National Laboratory Structural Biology Center beamlines, was supported by the U. S. Department of Energy, Office of Energy Research,. We acknowledge LS-CAT for time at APS Sector 32. Use of the National Magnetic Resonance Facility at Madison was supported in part by NIH grant RR02301 from the National Center for Research Resources (NCRR). This work is also based upon research conducted at the Northeastern Collaborative Access Team (NE-CAT) beamlines of the Advanced Photon Source, supported by NIH grant RR15301 from the NCRR. ‡ Atomic coordinates, along with structure factors for the X-ray structures and constraint lists for the NMR conformers, have been deposited in the Protein Data Bank, www.rcsb.org, under PDB # 2EVN (NMR structure of At1g77540), PDB # 1XMT (X-ray structure of At1g77540), and PDB # 2GDB (X-ray structure of At1g77540-CoA complex). Resonance assignments and primary NMR data have been deposited in BioMagResBank, www.bmrb.wisc.edu, under Arabidopsis thaliana At1g77540.1 for structural characterization on the basis of its target selection algorithm. This protein (At1g77540) consists of 103-residues (11.6 kDa) and had less than 25% sequence identity to any other structure deposited in the Protein Data Bank. In addition, no biochemical function had been established for this protein or its close homologs. However, the Superfamily server (1) predicted a very weak ...

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