Topology and secondary structure of the N-terminal domain of diacylglycerol kinase.
Prokaryotic diacylglycerol kinase (DAGK) functions as a homotrimer of 13 kDa subunits, each of which has three transmembrane segments. This enzyme is conditionally essential to some bacteria and serves as a model system for studies of membrane protein biocatalysis, stability, folding, and misfolding. In this work, the detailed topology and secondary structure of DAGK's N-terminus up through the loop following the first transmembrane domain were probed by NMR spectroscopy. Secondary structure was mapped by measuring 13C NMR chemical shifts. Residue-to-residue topology was probed by measuring 19F NMR relaxation rates for site-specifically labeled samples in the presence and absence of polar and hydrophobic paramagnetic probes. Most of DAGK's N-terminal cytoplasmic and first transmembrane segments are alpha-helical. The first and second transmembrane helices are separated by a short loop from residues 48 to 52. The first transmembrane segment extends from residues 32 to 48. Most of the N-terminal cytoplasmic domain lies near the interface but does not extend deeply into the membrane. Finally, catalytic activities measured for the single cysteine mutants before and after chemical labeling suggest that the N-terminal cytoplasmic domain likely contains a number of critical active site residues. The results, therefore, suggest that DAGK's active site lies very near to the water/bilayer interface.
Oxenoid
Kirill
K
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106-4970, USA.
Sönnichsen
Frank D
FD
Sanders
Charles R
CR
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000235:genetics; D019852:Diacylglycerol Kinase / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D005461:Fluorine; D019786:Gadolinium DTPA / Q000737:chemistry; D005960:Glucosides / Q000737:chemistry; D008823:Micelles; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D010713:Phosphatidylcholines / Q000737:chemistry; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D013816:Thermodynamics
D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D005960:Glucosides; D008823:Micelles; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D005461:Fluorine; C040358:dodecyl maltoside; D019852:Diacylglycerol Kinase; D019786:Gadolinium DTPA; D003545:Cysteine; C028694:1-palmitoyl-2-oleoylphosphatidylcholine