Abstract：
:Many viruses employ ATP-powered motors for genome packaging. We combined genetic, biochemical, and single-molecule techniques to confirm the predicted Walker-B ATP-binding motif in the phage λ motor and to investigate the roles of the conserved residues. Most changes of the conserved hydrophobic residues resulted in >107-fold decrease in phage yield, but we identified nine mutants with partial activity. Several were cold-sensitive, suggesting that mobility of the residues is important. Single-molecule measurements showed that the partially active A175L exhibits a small reduction in motor velocity and increase in slipping, consistent with a slowed ATP binding transition, whereas G176S exhibits decreased slipping, consistent with an accelerated transition. All changes to the conserved D178, predicted to coordinate Mg2+•ATP, were lethal except conservative change D178E. Biochemical interrogation of the inactive D178N protein found no folding or assembly defects and near-normal endonuclease activity, but a ∼200-fold reduction in steady-state ATPase activity, a lag in the single-turnover ATPase time course, and no DNA packaging, consistent with a critical role in ATP-coupled DNA translocation. Molecular dynamics simulations of related enzymes suggest that the aspartate plays an important role in enhancing the catalytic activity of the motor by bridging the Walker motifs and precisely contributing its charged group to help polarize the bound nucleotide. Supporting this prediction, single-molecule measurements revealed that change D178E reduces motor velocity without increasing slipping, consistent with a slowed hydrolysis step. Our studies thus illuminate the mechanistic roles of Walker-B residues in ATP binding, hydrolysis, and DNA translocation by this powerful motor.
journal_name
J Mol Bioljournal_title
Journal of molecular biologyauthors
delToro D,Ortiz D,Ordyan M,Pajak J,Sippy J,Catala A,Oh CS,Vu A,Arya G,Smith DE,Catalano CE,Feiss Mdoi
10.1016/j.jmb.2019.08.012subject
Has Abstractpub_date
2019-11-08 00:00:00pages
4455-4474issue
22eissn
0022-2836issn
1089-8638pii
S0022-2836(19)30521-2journal_volume
431pub_type
杂志文章abstract：:Carboxypeptidase M (CPM), an extracellular glycosylphosphatidyl-inositol(GPI)-anchored membrane glycoprotein belonging to the CPN/E subfamily of "regulatory" metallo-carboxypeptidases, specifically removes C-terminal basic residues from peptides and proteins. Due to its wide distribution in human tissues, CPM is belie...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2004.02.058
更新日期：2004-04-23 00:00:00
abstract：:Antibodies and prorenin mutants have long been used to structurally characterize prorenin, the inactive proenzyme form of renin. They were designed on the basis of homology models built using other aspartyl protease proenzyme structures since no structure was available for prorenin. Here, we present the first X-ray st...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2012.05.003
更新日期：2012-08-03 00:00:00
abstract：:Pax5 (paired box binding factor 5) is a critical regulator of transcription and lineage commitment in B lymphocytes. In B cells, mb-1 (Ig-alpha/immunoglobulin-associated alpha) promoter transcription is activated by Pax5 through its recruitment of E74-like transforming sequence (Ets) family proteins to a composite sit...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2009.07.028
更新日期：2009-09-18 00:00:00
abstract：:In all living cells, regulated passage across membranes of specific proteins occurs through a universally conserved secretory channel. In bacteria and chloroplasts, the energy for the mechanical work of moving polypeptides through that channel is provided by SecA, a regulated ATPase. Here, we use site-directed spin la...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2008.06.049
更新日期：2008-09-26 00:00:00
abstract：:Recombinant human interleukin-5 (rhIL-5) has been crystallized by the hanging drop vapor diffusion method using 0.1 M-Tris.HCl buffer (pH 8.5) containing 0.2 to 0.25 M-sodium acetate and 26 to 30% PEG 4000 at 22 degrees C. The parallel-piped crystals belong to the space group C2 with unit cell dimensions of a = 122.1 ...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1006/jmbi.1993.1110
更新日期：1993-02-20 00:00:00
abstract：:The pfkB gene of Escherichia coli is known to specify a minor phosphofructokinase, Pfk-2, in the wild-type strain; the pfkB1 mutation causes a 25-fold increase in the amount of Pfk-2 so that it adequately substitutes for mutational loss of the major phosphofructokinase, Pfk-1 (specified by pfkA); and another closely l...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/s0022-2836(83)80019-9
更新日期：1983-08-05 00:00:00
abstract：:A cytochrome c kinetic folding intermediate was studied by hydrogen exchange (HX) pulse labeling. Advances in the technique and analysis made it possible to define the structured and unstructured regions, equilibrium stability, and kinetic opening and closing rates, all at an amino acid-resolved level. The entire N-te...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2003.09.070
更新日期：2003-11-28 00:00:00
abstract：:We report the X-ray structure analysis and refinement at 1.9 A resolution of calf gamma-II crystallin, a lens-specific protein. The sequence of Croft (1972) has been modified to give a polypeptide chain of 174 residues (cf. 165). The protein has a symmetrical, hierarchical structure of two globular domains each compri...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/s0022-2836(83)80232-0
更新日期：1983-10-15 00:00:00
abstract：:NMR spectroscopy and simulated annealing calculations have been used to determine the three-dimensional structure of NaD1, a novel antifungal and insecticidal protein isolated from the flowers of Nicotiana alata. NaD1 is a basic, cysteine-rich protein of 47 residues and is the first example of a plant defensin from fl...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/s0022-2836(02)01103-8
更新日期：2003-01-03 00:00:00
abstract：:The process of heat denaturation of recombinant factor XIII (rFXIII), as well as its C-terminal 24 kDA and 12 kDa elastase-produced fragments starting at Ser514 and Thr628, respectively, was investigated in a wide range of conditions by fluorescence, CD and differential scanning calorimetry (DSC). It was found that th...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/s0022-2836(95)80060-3
更新日期：1995-04-28 00:00:00
abstract：:The mercury resistance locus encoded by Tn21 on the monocopy IncFII plasmid R100 (merTn21) consists of a metal-responsive activator/repressor, merR, which controls initiation of a polycistronic message that includes genes for the uptake (merTPC) and reduction (merA) of Hg2+ and merD, which may also play a minor regula...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/0022-2836(92)90919-b
更新日期：1992-05-20 00:00:00
abstract：:Chagasin is a protein produced by Trypanosoma cruzi, the parasite that causes Chagas' disease. This small protein belongs to a recently defined family of cysteine protease inhibitors. Although resembling well-known inhibitors like the cystatins in size (110 amino acid residues) and function (they all inhibit papain-li...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2007.05.005
更新日期：2007-08-03 00:00:00
abstract：:GDP liganded tubulin, which is inactive in microtubule assembly, polymerizes into rings more readily than the active GTP liganded protein. The structure of double rings made of highly purified GDP-tubulin has been characterized to 3 nm resolution with synchrotron X-ray solution scattering. The scattering profile has c...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1006/jmbi.1994.1282
更新日期：1994-04-29 00:00:00
abstract：:Dimers of the Rel/NFkappaB transcription factor family form with differential stabilities through the combinatorial association of five polypeptides: p50, p52, p65, cRel, and RelB. Here, we have characterized the nature of the monomer-dimer equilibrium of the p50 homodimer. Sedimentation equilibrium studies show that ...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1006/jmbi.1999.2823
更新日期：1999-06-18 00:00:00
abstract：:Crystal structures of Bacillus stearothermophilus TRS40 neopullulanase and its complexes with panose, maltotetraose and isopanose were determined at resolutions of 1.9, 2.4, 2.8 and 3.2A, respectively. Since the latter two carbohydrates are substrates of this enzyme, a deactivated mutant at the catalytic residue Glu35...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/s0022-2836(02)01402-x
更新日期：2003-02-07 00:00:00
abstract：:Human serum amyloid P component (SAP) is a normal plasma glycoprotein and the precursor of amyloid P component which is a universal constituent of the abnormal tissue deposits in amyloidosis. X-ray and neutron scattering data showed that pentameric or decameric ring structures for SAP in solution are readily distingui...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1006/jmbi.1997.1271
更新日期：1997-09-26 00:00:00
abstract：:We describe the physicochemical characterization of various circular and linear forms of the approximately 60 residue N-terminal Src homology 3 (SH3) domain from the murine c-Crk adapter protein. Structural, dynamic, thermodynamic, kinetic and biochemical studies reveal that backbone circularization does not prevent t...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1006/jmbi.2001.4631
更新日期：2001-05-18 00:00:00
abstract：:X-ray patterns have been obtained from solutions of microtubules and tubulin rings using synchrotron radiation. They are interpreted on the basis of the oriented fiber X-ray diagram of microtubules, and by comparing them with the calculated scattering traces of model structures. The data are consistent with models in ...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/s0022-2836(83)80040-0
更新日期：1983-06-15 00:00:00
abstract：:Signal transduction pathways mediated by cyclic-bis(3'→5')-dimeric GMP (c-di-GMP) control many important and complex behaviors in bacteria. C-di-GMP is synthesized through the action of GGDEF domains that possess diguanylate cyclase activity and is degraded by EAL or HD-GYP domains with phosphodiesterase activity. The...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2013.03.021
更新日期：2013-06-26 00:00:00
abstract：:Staphylococcus aureus is the major cause of nosocomial infections world-wide, with increasing prevalence of community-acquired diseases. The recent dramatic increase in multi-antibiotic resistance, including resistance to the last-resort drug, vancomycin, together with the lack of an effective vaccine highlight the ne...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2006.01.098
更新日期：2006-04-21 00:00:00
abstract：:A long-standing problem of molecular biology is the prediction of globular protein tertiary structure from the primary sequence. In the context of a new, 24-nearest-neighbor lattice model of proteins that includes both alpha and beta-carbon atoms, the requirements for folding to a unique four-member beta-barrel, four-...
journal_title：Journal of molecular biology
pub_type： 杂志文章,评审
doi：10.1016/0022-2836(91)80070-b
更新日期：1991-09-20 00:00:00
abstract：:An alphoid-like human repetitive DNA of the Sau3A family is present extrachromosomally and in the chromosomes. In the chromosomes, the DNA is located on chromosome 11 but related sequences are present in chromosome 17. We characterized the nature of the recombination involved in the excision of the extrachromosomal DN...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/0022-2836(87)90202-6
更新日期：1987-12-20 00:00:00
abstract：:Mutations in human cardiac myosin-binding protein C (cMyBP-C) gene are associated with familial hypertrophic cardiomyopathy (FHC), and most of them are predicted to produce COOH-truncated proteins. To understand the molecular mechanism(s) by which such mutations cause FHC, we analyzed (i) the accumulation of human cMy...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1006/jmbi.1999.3276
更新日期：1999-11-26 00:00:00
abstract：:The nine polyglutamine (polyQ) neurodegenerative diseases are caused in part by a gain-of-function mechanism involving protein misfolding, the deposition of β-sheet-rich aggregates and neuronal toxicity. While previous experimental evidence suggests that the polyQ-induced misfolding mechanism is context dependent, the...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2011.09.014
更新日期：2011-11-04 00:00:00
abstract：:Atomic positions obtained by X-ray crystallography are time and space averages over many molecules in the crystal. Importantly, interatomic distances, calculated between such average positions and frequently used in structural and mechanistic analyses, can be substantially different from the more appropriate time-aver...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2011.05.033
更新日期：2011-08-05 00:00:00
abstract：:Phage display, SELEX and other methods of combinatorial chemistry have become very popular means of finding ligands with high affinities to given targets. Despite their success, they suffer from numerous sources of error and bias, such as very low initial concentrations of species, non-specific binding, and the sampli...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1006/jmbi.1997.1555
更新日期：1998-04-10 00:00:00
abstract：:α-Synuclein (αSyn) is an intrinsically disordered protein that can form amyloid fibrils. Fibrils of αSyn are implicated with the pathogenesis of Parkinson's disease and other synucleinopathies. Elucidating the mechanism of fibril formation of αSyn is therefore important for understanding the mechanism of the pathogene...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2019.05.047
更新日期：2019-08-09 00:00:00
abstract：:Dimerization is indispensible for release of the human immunodeficiency virus protease (PR) from its precursor (Gag-Pol) and ensuing mature-like catalytic activity that is crucial for virus maturation. We show that a single-chain Fv fragment (scFv) of a previously reported monoclonal antibody (mAb1696), which recogniz...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2012.05.024
更新日期：2012-09-14 00:00:00
abstract：:Aminoglycosides are a medically important class of antibiotics used to treat serious infections. Methylation of the ribosomal target is an emerging mechanism that produces a high level of resistance to all clinically available aminoglycosides for systemic therapy except streptomycin. ArmA was the first methyltransfera...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2006.03.038
更新日期：2006-06-02 00:00:00
abstract：:Challenges in the production of integral membrane proteins for structural studies include low expression levels, incorrect membrane insertion, aggregation and instability. In this report, we describe a "funnel approach" to overcoming these difficulties and demonstrate its efficacy in a case study of 36 prokaryotic P-t...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2007.12.059
更新日期：2008-03-14 00:00:00
