Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductance regulator
2003; Springer Nature; Volume: 23; Issue: 2 Linguagem: Inglês
10.1038/sj.emboj.7600040
ISSN1460-2075
AutoresH.A. Lewis, Sean G. Buchanan, S.K. Burley, K. Conners, M. Dickey, Michael R. Dorwart, Richard Fowler, Xia Gao, William B. Guggino, Wayne A. Hendrickson, J.F. Hunt, M.C. Kearins, Don Lorimer, Peter C. Maloney, Kai Post, Kanagalaghatta R. Rajashankar, M. Rutter, J.M. Sauder, Stephanie Shriver, Patrick H. Thibodeau, Philip Thomas, Marie Zhang, Xun Zhao, Spencer Emtage,
Tópico(s)Respiratory viral infections research
ResumoArticle18 December 2003free access Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductance regulator Hal A Lewis Corresponding Author Hal A Lewis Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author Sean G Buchanan Sean G Buchanan Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author Stephen K Burley Stephen K Burley Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author Kris Conners Kris Conners Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author Mark Dickey Mark Dickey Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author Michael Dorwart Michael Dorwart Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Richard Fowler Richard Fowler Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author Xia Gao Xia Gao Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author William B Guggino William B Guggino Department of Physiology, School of Medicine, The Johns Hopkins University, Baltimore, MD, USA Search for more papers by this author Wayne A Hendrickson Wayne A Hendrickson Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University, New York, NY, USA Search for more papers by this author John F Hunt John F Hunt Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Margaret C Kearins Margaret C Kearins Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author Don Lorimer Don Lorimer Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author Peter C Maloney Peter C Maloney Department of Physiology, School of Medicine, The Johns Hopkins University, Baltimore, MD, USA Search for more papers by this author Kai W Post Kai W Post Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author Kanagalaghatta R Rajashankar Kanagalaghatta R Rajashankar Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author Marc E Rutter Marc E Rutter Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author J Michael Sauder J Michael Sauder Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author Stephanie Shriver Stephanie Shriver Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author Patrick H Thibodeau Patrick H Thibodeau Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Philip J Thomas Philip J Thomas Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Marie Zhang Marie Zhang Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author Xun Zhao Xun Zhao Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author Spencer Emtage Spencer Emtage Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author Hal A Lewis Corresponding Author Hal A Lewis Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author Sean G Buchanan Sean G Buchanan Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author Stephen K Burley Stephen K Burley Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author Kris Conners Kris Conners Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author Mark Dickey Mark Dickey Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author Michael Dorwart Michael Dorwart Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Richard Fowler Richard Fowler Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author Xia Gao Xia Gao Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author William B Guggino William B Guggino Department of Physiology, School of Medicine, The Johns Hopkins University, Baltimore, MD, USA Search for more papers by this author Wayne A Hendrickson Wayne A Hendrickson Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University, New York, NY, USA Search for more papers by this author John F Hunt John F Hunt Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Margaret C Kearins Margaret C Kearins Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author Don Lorimer Don Lorimer Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author Peter C Maloney Peter C Maloney Department of Physiology, School of Medicine, The Johns Hopkins University, Baltimore, MD, USA Search for more papers by this author Kai W Post Kai W Post Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author Kanagalaghatta R Rajashankar Kanagalaghatta R Rajashankar Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author Marc E Rutter Marc E Rutter Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author J Michael Sauder J Michael Sauder Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author Stephanie Shriver Stephanie Shriver Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author Patrick H Thibodeau Patrick H Thibodeau Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Philip J Thomas Philip J Thomas Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Marie Zhang Marie Zhang Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author Xun Zhao Xun Zhao Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author Spencer Emtage Spencer Emtage Structural GenomiX Inc., San Diego, CA, USA Search for more papers by this author Author Information Hal A Lewis 1, Sean G Buchanan1, Stephen K Burley1, Kris Conners1, Mark Dickey1, Michael Dorwart2, Richard Fowler1, Xia Gao1, William B Guggino3, Wayne A Hendrickson4, John F Hunt5, Margaret C Kearins1, Don Lorimer1, Peter C Maloney3, Kai W Post1, Kanagalaghatta R Rajashankar1, Marc E Rutter1, J Michael Sauder1, Stephanie Shriver1, Patrick H Thibodeau2, Philip J Thomas2, Marie Zhang1, Xun Zhao1 and Spencer Emtage1 1Structural GenomiX Inc., San Diego, CA, USA 2Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX, USA 3Department of Physiology, School of Medicine, The Johns Hopkins University, Baltimore, MD, USA 4Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University, New York, NY, USA 5Department of Biological Sciences, Columbia University, New York, NY, USA *Corresponding author. Structural GenomiX Inc., 10505 Roselle St., San Diego, CA 92121, USA. Tel.: +1 858 228 1555; Fax: +1 858 457 4533; E-mail: [email protected] The EMBO Journal (2004)23:282-293https://doi.org/10.1038/sj.emboj.7600040 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a chloride channel. Nucleotide-binding domain 1 (NBD1), one of two ABC domains in CFTR, also contains sites for the predominant CF-causing mutation and, potentially, for regulatory phosphorylation. We have determined crystal structures for mouse NBD1 in unliganded, ADP- and ATP-bound states, with and without phosphorylation. This NBD1 differs from typical ABC domains in having added regulatory segments, a foreshortened subdomain interconnection, and an unusual nucleotide conformation. Moreover, isolated NBD1 has undetectable ATPase activity and its structure is essentially the same independent of ligand state. Phe508, which is commonly deleted in CF, is exposed at a putative NBD1-transmembrane interface. Our results are consistent with a CFTR mechanism, whereby channel gating occurs through ATP binding in an NBD1–NBD2 nucleotide sandwich that forms upon displacement of NBD1 regulatory segments. Introduction Cystic fibrosis (CF) is the most prevalent lethal, autosomal-recessive genetic disease among Caucasians. CF patients have severely reduced life expectancies, largely because of chronic pulmonary damage. The root cause of CF is in defective cystic fibrosis transmembrane conductance regulator (CFTR) (Riordan et al, 1989). Human CFTR is a 1480 residue, multidomain, integral membrane protein that regulates chloride ion flow across the cell membrane. It is a member of the ATP-binding cassette (ABC) transporter superfamily of proteins and consists of two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory region (R) arranged in the order MSD1–NBD1–R–MSD2–NBD2 (Figure 1). The NBDs of ABC transporters are typified by a consensus ATP-binding region, which encompasses two Walker motifs (A and B regions), a highly conserved region called the signature sequence (LSGGQ), plus other conserved functional features identified as the Q- and H-loops, named respectively for glutamine and histidine residues involved in ATP recognition and hydrolysis. The most common CF mutation is the deletion of CFTR phenylalanine 508 (ΔF508), which is located in NBD1. In total, 70% of CF alleles have ΔF508 and 90% of CF patients have at least one copy of this deletion. A better understanding of the structure and function of NBD1 and the role of Phe508 may accelerate the development of new approaches to the treatment of CF. Figure 1.Domain organization of CFTR. The five domains of CFTR are shown. Also indicated is a putative nucleotide-binding domain association in which the ATP-binding site of one NBD is opposed by the signature sequence of the other NBD. Inactivity at the NBD1 ATP-binding site is indicated by Ser residues in place of the catalytic Glu and His in addition to His residues substituted for the Gln and central Gly in the NBD2 signature sequence. Download figure Download PowerPoint Atomic-level structural information has been obtained for components of several ABC transporter systems. Complete bacterial transmembrane transporter proteins MsbA (Chang and Roth, 2001; Chang, 2003) and BtuCD (Locher et al, 2002) have been analyzed at modest resolution, showing similar associations between NBD and MSD domains but markedly different overall architectures. High-resolution X-ray structures have also been determined for several prokaryotic NBDs (Schmitt and Tampe, 2002), most recently HlyB (Schmitt et al, 2003) and GlcV (Verdon et al, 2003), and for one eukaryotic NBD, TAP1 (Gaudet and Wiley, 2001). All such NBDs have a common fold characterized by two subdomains: one contains an F1-like ATP-binding core plus an ABC-specific antiparallel β region and the other an ABC-specific α-helical domain (Karpowich et al, 2001). The F1-like portion contains the primary determinants of nucleotide binding; the antiparallel β portion adds interactions to the base and ribose groups; and the ABC signature sequence of the α-helical domain from a dimer mate completes productive coordination of the ATP β- and γ-phosphate groups of the nucleotide (Figure 1). Typical ABC transporters are thought to function as labile dimers in which coupling of ATP hydrolysis to movements in transmembrane segments drives the translocation of relevant entities across the membrane. The Rad50 DNA repair enzyme, a remote homolog of ABC proteins, provided the first structural model for the dimer state (Hopfner et al, 2000), and similar dimers form in BtuCD (Locher et al, 2002) and in a catalytically impaired E171Q variant of MJ0796 when complexed with ATP (Smith et al, 2002). The nucleotides in this symmetric dimer are sandwiched at the interface between protomers such that the LSGGQ residues from one complete the binding interactions of nucleotide in the apposing protomer. The putative functional NBD 'dimer' in CFTR is believed to be intramolecular and necessarily asymmetric since 'hemichannel' constructs produced wild-type activity when expressed together but not separately (Ostedgaard et al, 1997; Chan et al, 2000). The boundaries of CFTR NBD1 have been a matter of some controversy. It has been suggested to begin in the span from residue 373 (Wang et al, 2002) to residue 441 (Bianchet et al, 1997) at the N-terminus and to end in the span from residue 586 (Riordan et al, 1989) to residue 684 (Bianchet et al, 1997) at the C-terminus (human CFTR numbering, used throughout this paper). A compelling functional definition based on the coexpression of severed and deleted CFTR constructs gave boundaries within 433–633 for NBD1 (Chan et al, 2000). Many different expression constructs have been used in studies of NBD1 function, sometimes yielding conflicting results. Indeed, several aspects of CFTR NBD1 function remain poorly characterized. The most important outstanding issues pertain to ATP binding and hydrolysis, conformational adaptability in the domain, the effects of NBD1 phosphorylation, and the structural consequences of disease-causing mutations, in particular ΔF508. We have determined the crystal structure of murine NBD1 to shed light on these vital mechanistic issues. Results mNBD1 expression and purification Given the uncertainty in the globular domain definition of CFTR NBD1, we chose to clone and express in Escherichia coli many constructs in parallel covering residues from 363 to 686. A pan-genomic approach was employed, testing the solubility of such NBD1 constructs from 10 organisms (human, mouse, baboon, macaque, sheep, rabbit, frog, salmon, killifish, and dogfish). The high-level production of soluble proteins (>5 mg/l) that are nonaggregated in solution was limited to a narrow globular domain definition (N-terminus: residues 385–391; C-terminus: residues 670–680). Optimal recombinant protein was obtained from mouse CFTR with an expression construct spanning residues 389–673. Attempts with human NBD1 have not yet succeeded. We refer to the resulting mouse proteins as mNBD1 and mNBD1-P for the unphosphorylated and phosphorylated forms, respectively. Dynamic light scattering (DLS) and analytical gel filtration chromatography (GFC) measurements indicated that purified, recombinant mNBD1 was both monodisperse and monomeric. This protein was used for all of the structural studies reported here. A mutated version, K464A, which was expected to have reduced ATP binding, was also cloned and purified in the same manner and was used for ATP-binding measurements (see below). mNBD1 crystallization and structure determination Both native (S-Met) and selenomethionyl (Se-Met) mNBD1 crystallized in two morphologies under similar conditions: parallelepipeds in space group P4212 and tetragonal bipyramids in space group I4122. The structure of mNBD1 bound to Mg-AMP.PNP was initially determined by multiple isomorphous replacement with anomalous scattering (MIRAS) phasing from Ta6Br12-soaked I4122 crystals. This was subsequently improved using single-wavelength anomalous diffraction (SAD) data from Se-Met mNBD1 P4212 crystals. Most of the protein sequence could be built into the SAD-generated experimental electron density map, the primary exception being residues 413–428, which were not well visualized (exact boundaries are given in Table 1 for each refined molecule). The initial structure of mNBD1 was subsequently used to solve the I4122 structure in the presence of Mg-ATP by molecular replacement and to determine isomorphous P4212 structures in the presence of Mg-ATP, Mg-ADP, and in the absence of nucleotide. The conformation of mNBD1 is essentially the same in all crystal forms irrespective of the identity of the bound nucleotide and also in each of the four copies in the P4212 crystals and two copies in the I4122 crystal. Each mNBD1 molecule is associated in a four-fold symmetric, head-to-tail ring structure that recurs three times in the P4212 lattice and once in the I4122 lattice. Table 1. Data collection and refinement statistics Space group Resolution (Å) Nucleotide Completeness (%) Rsym (%) Redundancy I/σ(I) (overall/outer shell) (overall/outer shell) (overall/outer shell) (overall/ outer shell) mNBD1 data collection mNBD1+AMP.PNP P4212 33.0–2.50 AMP.PNP 99.9/99.6 12.2/35 14.1/13.6 5.4/1.8 mNBD1-P+ATP P4212 24.0–2.35 ATP 97.6/92.4 9.0/46 4.6/2.4 14.7/2.1 mNBD1 apo P4212 38.9–2.20 None 93.2/95.1 7.6/44 5.1/4.1 19.8/3.1 mNBD1+ATP P4212 39.2–2.20 ATP 98.5/92.7 7.3/50 7.0/5.7 27.7/4.0 mNBD1+ADP P4212 36.5–2.55 ADP 100.0/99.4 7.7/35 9.8/9.9 8.0/1.9 mNBD1+ATP (2) I4122 49.7–3.00 ATP 100.0/100.0 7.3/98 8.2/8.3 27.6/2.0 Resolution (Å) R Rfree Waters RMSD bond length (Å) RMSD bond angles (°) Average B-factors (Å2) Refinement statistics mNBD1+AMP.PNP (1Q3 H) 33.0–2.50 0.215 0.266 658 0.008 1.9 36.4 mNBD1-P+ATP (1R0Z) 24.0–2.35 0.221 0.258 304 0.015 1.5 48.0 mNBD1 apo (1R0W) 38.9–2.20 0.231 0.262 423 0.021 2.0 30.8 mNBD1+ATP (1R0X) 39.2–2.20 0.234 0.266 378 0.021 2.0 39.1 mNBD1+ADP (1R0Y) 36.0–2.55 0.207 0.257 195 0.015 1.7 45.6 mNBD1+ATP (2) (1R10) 30.0–3.00 0.228 0.265 0 0.012 1.3 80.6 Ramachandran distribution Molecule A Molecule B Molecule C Molecule D Core Allowed Disallowed Residues modeled mNBD1+AMP.PNP 390–412, 429–670 389–412, 429–670 388–412, 429–670 390–412,429–670 92.0% 7.9% 0.0% mNBD1-P+ATP 390–413, 420–670 389–412, 420–670 388–413, 420–670 390–407, 430–670 91.2% 8.7% 0.1% mNBD1 apo 390–413, 429–670 389–413, 429–670 388–413, 430–670 390–412, 430–670 92.4% 7.5% 0.1% mNBD1+ATP 390–411, 429–670 389–411, 429–670 388–411, 430–670 390–411, 430–670 91.2% 8.7% 0.0% mNBD1+ADP 388–413, 429–671 388–411, 428–670 388–412, 430–670 390–412, 430–671 89.9% 9.9% 0.1% mNBD1+ATP (2) 391–412, 429–670 391–412, 429–670 — — 85.5% 14.5% 0.0% Rsym=ΣhklΣi∣Ii(hkl)−〈I(hkl)〉∣/ΣhklΣiIi(hkl), where Ii is the intensity of the observation and 〈I〉 is the mean intensity of the reflection. R=Σ∣∣Fo∣−∣Fc∣∣/Σ∣Fo∣, where Fo and Fc are the observed and calculated structure-factor amplitudes. Rfree is the same as R except calculated for 5% of the data randomly omitted from the refinement. RMSD is root-mean-square deviation. Average B-factors are reported for all nonhydrogen atoms. The values in parenthesis following the dataset names are the Protein Data Bank identifiers for the respective structures. Overall structure of CFTR NBD1 The mNBD1 domain has a core tertiary structure similar to NBDs from other ABC transporters, but this core is modified with major additions and deletions. Figure 2B shows a topology diagram of mNBD1, indicating through color coding the subdomains and those regions of mNBD1 that show significant differences from other ABC structures. Secondary structural elements in common with most known ABC structures are given conventional designations (S1, S2, S3, H1, etc.) and additional elements found in mNBD1 are denoted with lowercase letters (H1b, H1c, S6b, etc.). The three-dimensional course of the polypeptide chain is shown as a ribbon diagram in Figure 3A, where the structural elements are colored in the same code as in Figure 2B and key elements in the binding of ATP are also identified. Figure 3B shows a worm diagram of mNBD1 in which the thickness of the trace is proportional to the B-factors of the Cα atoms, thereby reflecting potential mobility of the polypeptide backbone. Regions of highest mobility are near the N- and C-termini and at the inserted and partially disordered segment between S1 and S2. B-factors are commonly elevated near termini and in some loops, but there may be special relevance here in relation to the putative NBD1/NBD2 interface (see below). Figure 2.Sequence alignment and topology diagram of mNBD1. (A) Sequence alignment of human and mouse NBD1 and NBD2 with NBD domains from other ABC transporters. A blue background indicates β-strands while pink indicates α-helices in known structures. Solid circles mark the locations of common CF-causative mutations, solid triangles the locations of deletion mutations, and P indicates where phosphorylation by PKA was observed in the mNBD1–P structure. Residues with high sequence conservation in ABC domains are highlighted in blue bold font. Red bold font indicates residues that significantly vary from this conservation. The secondary structure of mNBD1 is indicated graphically above the residue numbering row and is color coded by subdomain as in Figure 2B. (B) Topology diagram of mNBD1. The F1-type ATP-binding core subdomain is shown in gold, the ABC α-subdomain in cyan, and the ABC β-subdomain in green. Regions of mNBD1 that are different from previous ABC structures are shown in gray. Circles indicate the positions of 310 helices. Download figure Download PowerPoint Figure 3.Structural fold of mNBD1. (A) Stereo ribbon diagram of mNBD1. ATP is shown in ball and stick representation. The subdomains are color coded as in Figure 2B. The dotted red line indicates residues missing from the mNBD1 model. (B) Stereo worm diagram of mNBD1. The worm thickness is indicative of the relative B-factor of the residues ranging from 18 Å2 (thinnest) to 65 Å2 (thickest). Color coding of subdomains is according to Figure 2B. (C) Stereo Cα diagram of the superposition of representative ABC domain structures onto mNBD1. TAP1 (thin red), MJ0796 (thin green), and HisP (thin yellow) structures superimposed onto mNBD1 (thick blue). Superposition based on least-squares alignments of the F1-type core and ABC-specific antiparallel β-subdomains. (D) Backbone structure of mNBD1 illustrating positions of phosphorylation and CF-causative mutations. Left: mNBD1 is seen in the same orientation and subdomain colorization as in Figure 3A. Right: the same structure rotated 80° toward the viewer. Helices of regulatory segments are drawn as ribbons; the remaining polypeptide chain is a worm drawing. ATP is shown as ball and stick. Ser422, Ser659, Ser660, and Ser670 side chains are shown in purple. Residues 420–428 become ordered upon phosphorylation (solid red). The remaining residues of the structure that were not modeled (414–419) are indicated as red dots. Side chains are shown at sites of common CF-causative mutations (Ala455, Gly480, Ile506, Ile507, Ser549, Gly551, Ala559, Arg560, Tyr569, and Asp648 colored yellow; Phe508 in green). The di-acidic code residues (D565 and D567) are in gold. Download figure Download PowerPoint The core structure of mNBD1-ATP most closely resembles that of TAP1-ADP with a root-mean-square deviation (r.m.s.d.) of 1.9 Å for 176 Cα atoms spanning a region with 26% sequence identity. MJ0796-ATP is next closest overall (2.4 Å r.m.s.d. over the same span; 30% sequence identity). This alignment omits the regions between S1 and S2, between S4 and S6, and beyond S10, which exhibit conformation differences between ABC structures. Figure 2A presents a structure-based sequence alignment of mNBD1 with the ABC domains of some known structures and with selected sequences from human ABC transporters, including human CFTR. These comparisons should help to clarify the discussions of CFTR function. Four major structural features distinguish mNBD1 from other ABC NBDs. This is evident in Figure 3C, where mNBD1 is superimposed onto three representative ABC structures. First, mNBD1 contains an insertion of about 35 residues between β-strands S1 and S2. This insertion is composed of two short α-helices (denoted H1b and H1c) separated by a flexible linker region that was not observed in the electron density map (residues 413–428, red dotted line in Figure 3A). The N-terminal β-strand S1 is common to all ABC domain structures, and a conserved aromatic residue near its C-terminus (corresponding to Trp401 in CFTR) stacks against the adenine base in previously reported structures of ABC domains complexed with nucleotides. The insertion leads to an altered binding geometry for the base and ribose in mNBD1 and includes a segment (415–432) that can be deleted while preserving function (Chan et al, 2000). Second, mNBD1 lacks a 14–27 residue region between β-strands S4 and S6 (residues 485–488) that typically contains an additional β-strand and an α-helix (denoted S5 and H2 in Figure 2A; located lower left in Figure 3C). Third, relative to the prokaryotic ABC structures, mNBD1 is truncated between helices H3 and H4 (lower right of Figure 3C). Finally, the C-terminus of mNBD1 includes a long α-helix (H9b) that is not present in other ABC domain structures. The hydrophobic face of this amphipathic α-helix packs against a hydrophobic surface formed primarily by the preceding α-helix H9. The H9b segment of the polypeptide chain has been considered to be part of the R domain as it contains two potentially regulatory phosphorylation sites, Ser660 and Ser670 (Chen et al, 2000). However, the structure and our expression experiments lead us to think of H9b as an integral component of the mNBD1 fold; at a minimum, the association is favorable. Preparation, crystallization, and structure determination of triphospho-mNBD1 Phosphorylation of CFTR, particularly in the R domain, is thought to regulate channel opening and closing, and protein kinase A (PKA) plays an important role in this process (Ostedgaard et al, 2001). There are 21 PKA phosphorylation motifs (Kennelly and Krebs, 1991) in human CFTR. About one-quarter of these putative phosphorylation sites are located in mNBD1 (Ser422, Ser489, Ser519, Ser557, Ser660, and Ser670), and the murine sequence presents an additional PKA site at Ser659. To identify CFTR NBD1 residues available for PKA phosphorylation in vitro, we incubated mNBD1 with PKA and analyzed the results using phosphopeptide mapping by mass spectrometry. Residues Ser422, Ser659, Ser660, and Ser670 were seen to be phosphorylated with Ser659 to a lesser extent of only about one-quarter. The phosphorylated form of the protein, mNBD1-P, crystallized isomorphously with the unmodified protein and we were able to determine its structure. Those portions of mNBD1 that are well-ordered in the unphosphorylated state are unchanged in the mNBD1-P structure, but the presence of phosphorserine 422 confers order on residues 420–428 (solid red ribbon segment in Figure 3D) in the inserted loop where a phosphate group is visible on the side chain of Ser422. In addition, phosphorylation is evident at residues Ser660 and Ser670, and partially at Ser659 (only seen in molecule A in the asymmetric unit). Phosphorylation at Ser422 (Chang et al, 1993), Ser660 (Cheng et al, 1991; Winter and Welsh, 1997), and Ser670 (Wilkinson et al, 1997) has been observed in human CFTR and each has been shown to have a specific effect on the activity of CFTR. Based on this evidence as well as discussions on the relevance of these regions to a putative NBD1–NBD2 heterodimer (see below), we will refer to the S1–S2 loop and H9c structures as the regulatory insertion and regulatory extension, respectively. Nucleotide coordination in mNBD1 crystal structures Nucleotides in the various crystal complexes with mNBD1 (Table 1) are all bound in a very similar manner. This mode of binding has aspects in common with that in other ABC transporter NBDs, but the comparison is distinguished as much by differences as similarities. Since Mg-ATP has its γ-phosphate intact when cocrystallized with mNBD1, whether phosphorylated or not, mNBD1 is not an active ATPase as is typical for such domains. Moreover, since the protein conformation is the same when uncomplexed or complexed with ADP as when complexed with ATP (r.m.s.d.=0.2–0.3 Å), the possibility is raised that nucleotide binding to the NBD1 site in CFTR is not involved directly in movements thought to drive ABC transporter action in other cases (Yuan et al, 2001). Canonical features of nucleotide binding in mNBD1 all involve interactions with the phosphate groups (Figure 4A). Specifically, Lys464 and Thr465 (Walker A), Asp572 (Walker B), and Gln493 (Q-loop) hydrogen bond with the phosphates and/or coordinate the Mg2+ ion as in other ABC domain structures. On the other hand, consistent with a catalytically inactive site, there are also significant differences. Namely, the Walker-B carboxylate residue, typically glutamate, that serves as the catalytic base in active ABC transporters (Moody et al, 2002) is replaced by serine in NBD1 (Ser573), and the canonical H-loop histidine residue becomes Ser605. Both of these fail to hydrogen bond with the γ-phosphate as in related complexes (Hung et al, 1998; Smith et al, 2002). Figure 4.Close-up on ATP binding by mNBD1. (A) Canonical hydrogen bonding interactions in Mg-ATP mNBD1. Some relevant hydrogen bonds are indicated as green lines. Some residues in the foreground and background have been removed to clarify the interactions, here and in (B) and (C). (B) Differences in adenine base recognition from other ABC domains. Left: adenine stacks against Tyr11 of MJ0796 (PDB ID code 1L2T). Right: adenine of ATP makes edge-to-face interactions with Phe430 of mNBD1. (C) Added structure in phosphorylated mNBD1. The ATP molecule plus magnesium in blue, the additional mNBD1 residues observed in the phosphorylated state in white, the phosphate atoms of Ser422 and Ser660 in
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