Crystal structure of E. coli Hsp100 ClpB nucleotide-binding domain 1 (NBD1) and mechanistic studies on ClpB ATPase activity.
E. coli Hsp100 ClpB was recently identified as a critical part in a multi-chaperone system to play important roles in protein folding, protein transport and degradation in cell physiology. ClpB contains two nucleotide-binding domains (NBD1 and NBD2) within their primary sequences. NBD1 and NBD2 of ClpB can be classified as members of the large ATPase family known as ATPases associated with various cellular activities (AAA). To investigate how ClpB performs its ATPase activities for its chaperone activity, we have determined the crystal structure of ClpB nucleotide-binding domain 1 (NBD1) by MAD method to 1.80 A resolution. The NBD1 monomer structure contains one domain that comprises 11 alpha-helices and six beta-strands. When compared with the typical AAA structures, the crystal structure of ClpB NBD1 reveals a novel AAA topology with six-stranded beta-sheet as its core. The N-terminal portion of NBD1 structure has an extra beta-strand flanked by two extra alpha-helices that are not present in other AAA structures. Moreover, the NBD1 structure does not have a C-terminal helical domain as other AAA proteins do. No nucleotide molecule is bound with ClpB NBD1 in the crystal structure probably due to lack of the C-terminal helix domain in the structure. Isothermal titration calorimetry (ITC) studies of ClpB NBD1 and other ClpB deletion mutations showed that either ClpB NBD1 or NBD2 alone does not bind to nucleotides. However, ClpB NBD2 combined with ClpB C-terminal fragment can interact with one ADP or ATP molecule. ITC data also indicated that full-length ClpB could bind two ADP molecules or one ATP analogue ATPgammaS molecule. Further ATPase activity studies of ClpB and ClpB deletion mutants showed that only wild-type ClpB have ATPase activity. None of ClpB NBD1 domain, NBD2 domain and NBD2 with C-terminal fragment has detectable ATPase activities. On the basis of our structural and mutagenesis data, we proposed a "see-saw" model to illustrate the mechanisms by which ClpB performs its ATPase activities for chaperone functions.
Li
Jingzhi
J
Department of Cell Biology, Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, 35294-005, USA.
Sha
Bingdong
B
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D003460:Crystallization; D018360:Crystallography, X-Ray; D049071:Endopeptidase Clp; D004926:Escherichia coli / Q000201:enzymology*; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D015800:Protozoan Proteins / Q000737:chemistry* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D015800:Protozoan Proteins; D000255:Adenosine Triphosphate; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C095049:ClpB protein, Leishmania