J Michael Sauder - Structure and dynamics of NBD1 from CFTR characterized using crystallography and hydrogen/deuterium exchange mass spectrometry

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  Publication Details (including relevant citation   information):

  J Mol Biol (2010) 396: 406-430.

  Lewis HA, Wang C, Zhao X, Hamuro Y, Conners K, Kearins MC, Lu F,   Sauder JM, Molnar KS, Coales SJ, Maloney PC, Guggino WB, Wetmore   DR, Weber PC, Hunt JF


  The DeltaF508 mutation in nucleotide-binding domain 1 (NBD1) of   the  cystic fibrosis transmembrane conductance regulator   (CFTR) is the  predominant cause of cystic fibrosis.   Previous biophysical studies on  human F508 and DeltaF508   domains showed only local structural changes  restricted to   residues 509-511 and only minor differences in folding  rate   and stability. These results were remarkable because DeltaF508   was  widely assumed to perturb domain folding based on the   fact that it  prevents trafficking of CFTR out of the   endoplasmic reticulum. However,  the previously reported   crystal structures did not come from matched  F508 and   DeltaF508 constructs, and the DeltaF508 structure contained    additional mutations that were required to obtain sufficient   protein  solubility. In this article, we present additional   biophysical studies  of NBD1 designed to address these   ambiguities. Mass spectral  measurements of backbone amide   (1)H/(2)H exchange rates in matched F508  and DeltaF508   constructs reveal that DeltaF508 increases backbone    dynamics at residues 509-511 and the adjacent protein segments   but not  elsewhere in NBD1. These measurements also confirm   a high level of  flexibility in the protein segments   exhibiting variable conformations in  the crystal   structures. We additionally present crystal structures of a    broader set of human NBD1 constructs, including one harboring   the  native F508 residue and others harboring the DeltaF508   mutation in the  presence of fewer and different   solubilizing mutations. The only  consistent conformational   difference is observed at residues 509-511.  The side chain   of residue V510 in this loop is mostly buried in all    non-DeltaF508 structures but completely solvent exposed in all   DeltaF508  structures. These results reinforce the   importance of the perturbation  DeltaF508 causes in the   surface topography of NBD1 in a region likely to  mediate   contact with the transmembrane domains of CFTR. However,   they  also suggest that increased exposure of the 509-511   loop and increased  dynamics in its vicinity could promote   aggregation in vitro and aberrant  intermolecular   interactions that impede trafficking in vivo.

  Address (URL): http://www.ncbi.nlm.nih.gov/pubmed/19944699