What the structure of a calcium pump tells us about its mechanism

2001 ◽  
Vol 356 (3) ◽  
pp. 665-683 ◽  
Author(s):  
Anthony G. LEE ◽  
J. Malcolm EAST
Keyword(s):  
Crystal Structure ◽  
Binding Sites ◽  
Calcium Pump ◽  
Transmembrane Region ◽  
Site Model ◽  
High Affinity ◽  
Cytoplasmic Surface ◽  
Α Helix ◽  
Access Pathway ◽  
Phosphorylation Domain

The report of the crystal structure of the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum in its Ca2+-bound form [Toyoshima, Nakasako and Ogawa (2000) Nature (London) 405, 647–655] provides an opportunity to interpret much kinetic and mutagenic data on the ATPase in structural terms. There are no large channels leading from the cytoplasmic surface to the pair of high-affinity Ca2+ binding sites within the transmembrane region. One possible access pathway involves the charged residues in transmembrane α-helix M1, with a Ca2+ ion passing through the first site to reach the second site. The Ca2+-ATPase also contains a pair of binding sites for Ca2+ that are exposed to the lumen. In the four-site model for transport, phosphorylation of the ATPase leads to transfer of the two bound Ca2+ ions from the cytoplasmic to the lumenal pair of sites. In the alternating four-site model for transport, phosphorylation leads to release of the bound Ca2+ ions directly from the cytoplasmic pair of sites, linked to closure of the pair of lumenal binding sites. The lumenal pair of sites could involve a cluster of conserved acidic residues in the loop between M1 and M2. Since there is no obvious pathway from the high-affinity sites to the lumenal surface of the membrane, transport of Ca2+ ions must involve a significant change in the packing of the transmembrane α-helices. The link between the phosphorylation domain and the pair of high-affinity Ca2+ binding sites is probably provided by two small helices, P1 and P2, in the phosphorylation domain, which contact the loop between transmembrane α-helices M6 and M7.

scholarly journals Characterization of inositol 1,4,5-trisphosphate- and inositol 1,3,4,5-tetrakisphosphate-binding sites in rat cerebellum

1991 ◽  
Vol 274 (3) ◽  
pp. 861-867 ◽  
Author(s):  
R A J Challiss ◽  
A L Willcocks ◽  
B Mulloy ◽  
B V L Potter ◽  
S R Nahorski
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Radioligand Binding ◽  
Specific Binding ◽  
Binding Activity ◽  
Inositol Polyphosphate ◽  
Homogeneous Population ◽  
Site Model ◽  
High Affinity ◽  
Pentosan Polysulphate

1. The properties of specific Ins(1,4,5)P3- and Ins(1,3,4,5)P4-binding sites have been compared in a crude ‘P2’ cerebellar membrane fraction. 2. A homogeneous population of [3H]Ins(1,4,5)P3-binding sites was present (KD 23.1 +/- 3.6 nM) at high density (Bmax. 11.9 +/- 1.8 pmol/mg of protein); whereas data obtained for [32P]Ins(1,3,4,5)P4 specific binding were best fitted to a two-site model, the high-affinity binding component (KD 2.6 +/- 0.7 nM) constituted 64.2 +/- 4.3% of the total population and was present at relatively low density (Bmax. 187 +/- 27 fmol/mg of protein). 3. The two high-affinity inositol polyphosphate-binding sites exhibited markedly different pH optima for radioligand binding, allowing the two sites to be independently investigated. At pH 8.0, [3H]Ins(1,4,5)P3 binding was maximal, whereas [32P]Ins(1,3,4,5)P4 specific binding was very low; conversely, at pH 5.0, [32P]Ins(1,3,4,5)P4 binding was maximal, whereas [3H]Ins(1,4,5)P3 binding was undetectably low. 4. Both inositol polyphosphate-binding sites exhibited marked positional and stereo-specificity. Of the analogues studied, only phosphorothioate substitution to form inositol 1,4,5-trisphosphorothioate was tolerated at the Ins(1,4,5)P3-binding site, with only a 2-3-fold loss of binding activity. Addition of a glyceroyl moiety at the 1-phosphate position or addition of further phosphate substituents at the 3- or 6-positions caused dramatic losses in displacing activity. Similarly, complete phosphorothioate substitution of Ins(1,3,4,5)P4 caused an approx. 6-fold loss of binding activity at the [32P]Ins(1,3,4,5)P4-binding site, whereas Ins(1,4,5,6)P4, Ins(1,3,4,6)P4, Ins(1,4,5)P3 and Ins(1,3,4,5,6)P5 were bound at least 100-fold weaker at this site. Therefore, only the phosphorothioate derivatives retained high affinity and selectivity for the two inositol polyphosphate-binding sites. 5. Heparin and pentosan polysulphate were potent but non-selective inhibitors at Ins(1,4,5)P3- and Ins(1,3,4,5)P4-binding sites. N-Desulphation (with or without N-reacetylation) of heparin decreased inhibitory activity at the Ins(1,4,5)P3-, but not at the Ins(1,3,4,5)P4-binding site; however, the selectivity of this effect was only about 4-fold. O- and N-desulphated N-reacetylated heparin was essentially inactive at both sites. 6. The results are discussed with respect to the separate identities of the inositol polyphosphate-binding sites.

scholarly journals High Affinity Agonistic Metal Ion Binding Sites within the Melanocortin 4 Receptor Illustrate Conformational Change of Transmembrane Region 3

2003 ◽  
Vol 278 (51) ◽  
pp. 51521-51526 ◽  
Author(s):  
Malin C. Lagerström ◽  
Janis Klovins ◽  
Robert Fredriksson ◽  
Davids Fridmanis ◽  
Tatjana Haitina ◽  
...  
Keyword(s):  
Conformational Change ◽  
Binding Sites ◽  
Metal Ion ◽  
Ion Binding ◽  
Metal Ion Binding ◽  
Transmembrane Region ◽  
High Affinity ◽  
Melanocortin 4 Receptor ◽  
Ion Binding Sites ◽  
Metal Ion Binding Sites

Pancreatic receptors for cholecystokinin: evidence for three receptor classes

1990 ◽  
Vol 258 (1) ◽  
pp. G86-G95 ◽  
Author(s):  
D. H. Yu ◽  
S. C. Huang ◽  
S. A. Wank ◽  
S. Mantey ◽  
J. D. Gardner ◽  
...  
Keyword(s):  
Binding Sites ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Future Studies ◽  
Site Model ◽  
High Affinity ◽  
The Past ◽  
Relative Potencies ◽  
Inhibition Curve

For inhibition of binding of 125I-Bolton-Hunter-labeled cholecystokinin octapeptide (125I-BH-CCK-8) to guinea pig pancreatic acini, the potencies for agonists were CCK-8 greater than desulfated [des(SO3)] CCK-8 greater than gastrin-17-I greater than pentagastrin greater than CCK-4 and for the antagonists L 364718 greater than proglumide analogue 10 greater than CBZ-CCK-(27-32)-NH2. For all non-sulfated agonists, the curves were biphasic with 20% of the tracer bound to sites with high affinity for these agonists with the following relative potencies: gastrin-17-I greater than pentagastrin greater than des(SO3)CCK-8 much greater than CCK-4; whereas 80% was bound to low-affinity sites with the following potencies: des(SO3)CCK-8 greater than gastrin-17-I = pentagastrin much greater than CCK-4. For L 364718 and proglumide analogue 10, 80% of 125I-BH-CCK-8 was bound to sites with high affinity for these antagonists and 20% to sites with low affinity. Analysis of the dose-inhibition curve for CCK-8 demonstrated two binding sites; however, comparison with the analysis in the presence of 0.1 microM gastrin-17-I suggested three binding sites. The gastrin-17-I dose-inhibition curve was significantly better fit by a three-site model than by a two-site model. The affinities of the various agonists and antagonists for the three sites were compared with their abilities to inhibit binding of 125I-gastrin-I and either stimulate or inhibit CCK-8-stimulated amylase release. These results demonstrate that 125I-BH-CCK-8 binds to three classes of receptors, not two as reported previously. Two classes are CCK-preferring, bind 83% of 125I-BH-CCK-8 at tracer concentrations, and comprise high- and low-affinity CCK-preferring sites that can be distinguished by all agonists but have equally high affinity for L 364718 and proglumide 10. A third class binds 17% of the tracer, cannot be differentiated from high-affinity CCK-preferring receptors by CCK-8, and has low affinities for L 364718 and proglumide 10. Future studies relating binding of 125I-BH-CCK-8 to biological activity or characterization of the CCK receptor by using radiolabeled agonists should consider CCK interaction with three receptors, not two as was done in the past.

Multiple high-affinity binding sites for

1996 ◽  
Vol 199 (11) ◽  
pp. 2429-2435 ◽  
Author(s):  
S Winberg ◽  
G Nilsson
Keyword(s):  
Binding Sites ◽  
Arctic Charr ◽  
G Protein Coupled Receptors ◽  
Receptor Ligands ◽  
Site Model ◽  
High Affinity ◽  
Saturation Binding ◽  
High Affinity Site ◽  
G Protein Coupled ◽  
Binding Curve

Binding of [3H]serotonin (5-HT) to membranes prepared from Arctic charr brain homogenates was most consistent with a one-site model for [3H]5-HT binding, with KD and Bmax values of 5.7±0.3 nmol l-1 and 60.7±7.3 fmol mg-1 protein, respectively. Similarly, 5-HT displacement of [3H]5-HT was best explained by a monophasic model with an apparent Ki of 4.3±0.7 nmol l-1. The ability of a number of synthetic 5-HT receptor ligands to displace [3H]5-HT was studied. 8OH-DPAT was found to interact with three [3H]5-HT binding sites, whereas buspirone, TFMPP, spiperone and mianserin all distinguish two sites. In the presence of 300 nmol l-1 buspirone, 8OH-DPAT and mianserin distinguished two [3H]5-HT binding sites, whereas spiperone interacted with only one. Moreover, 8OH-DPAT differentiated three [3H]5-HT binding sites even in the presence of 0.5 mmol l-1 GTP, making it unlikely that these sites represent different affinity states of G-protein-coupled receptors. GTP had no effect on apparent Ki values for 8OH-DPAT, but reduced the Bmax value of the high-affinity site by 60 %. GTP had a similar effect on the saturation binding curve for [3H]5-HT, reducing Bmax by approximately 50 %, whereas KD was unaffected. The results provide evidence for at least three different high-affinity [3H]5-HT binding sites, one of them showing a pharmacological profile strikingly similar to that of the mammalian 5-HT1A receptor.

scholarly journals A Spectroscopic Study on PtCl4(2−) Binding to Rabbit Skeletal Muscle G-Actin

1995 ◽  
Vol 2 (3) ◽  
pp. 127-136 ◽  
Author(s):  
Juan Zou ◽  
Hong-Ye Sun ◽  
Kui Wang
Keyword(s):  
Conformational Changes ◽  
Binding Sites ◽  
Binding Constant ◽  
Intrinsic Fluorescence ◽  
Molar Ratio ◽  
Second Step ◽  
High Affinity ◽  
Α Helix ◽  
Discontinuous Mode ◽  
Sulfur Containing

It was found that the binding of PtCl42− to G-actin and the consequent conformational changes are different with those for hard acids. It is a two-step process depending on molar ratio PtCl42−/actin (R). In the first step, R less than 25, the PtCl42− ions are bound to sulfur-containing groups preferentially. These high-affinity sites determined by Scatchard approach are characterized by n1 = 30 with average binding constant K1=1.0×107M-1. The conformational changes are significant as characterized by N-(1-pyrenyl) maleimide(NPM) labeled fluorescence, intrinsic fluorescence and CD spectra. EPR spectroscopy of maleimide spin labeled(MSL) actin demonstrated that even PtCl42−binding is limited to a very small fraction of high-affinity sites(R<1), it can bring about a pronounced change of conformation. In the range of R=25-40, high-affinity sites accessible are saturated. In the second step(R>40) , deep-buried binding sites turn out to be accessible as a result of the accumulated conformational changes. These new binding sites are estimated to be n2=26 with average binding constant K2=2.1×106M-1. Although in this step the quenching of intrinsic fluorescence goes on and the NPM-labled thiols moves to more hydrophilic environment, no change in α-helix content was found. These results suggested that with increasing in PtCl42− binding, the G-actin turns to an open and loose structure in a discontinuous mode.

scholarly journals What ATP binding does to the Ca2+pump and how nonproductive phosphoryl transfer is prevented in the absence of Ca2+

2020 ◽  
Vol 117 (31) ◽  
pp. 18448-18458 ◽  
Author(s):  
Yoshiki Kabashima ◽  
Haruo Ogawa ◽  
Rie Nakajima ◽  
Chikashi Toyoshima
Keyword(s):  
Crystal Structure ◽  
Cardiac Muscle ◽  
Binding Sites ◽  
Phosphorylation Site ◽  
Phosphoryl Transfer ◽  
Atp Binding ◽  
Physiological Conditions ◽  
High Affinity ◽  
Ph Values ◽  
P Domain

Under physiological conditions, most Ca2+-ATPase (SERCA) molecules bind ATP before binding the Ca2+transported. SERCA has a high affinity for ATP even in the absence of Ca2+, and ATP accelerates Ca2+binding at pH values lower than 7, where SERCA is in the E2 state with low-affinity Ca2+-binding sites. Here we describe the crystal structure of SERCA2a, the isoform predominant in cardiac muscle, in the E2·ATP state at 3.0-Å resolution. In the crystal structure, the arrangement of the cytoplasmic domains is distinctly different from that in canonical E2. The A-domain now takes an E1 position, and the N-domain occupies exactly the same position as that in the E1·ATP·2Ca2+state relative to the P-domain. As a result, ATP is properly delivered to the phosphorylation site. Yet phosphoryl transfer never takes place without the filling of the two transmembrane Ca2+-binding sites. The present crystal structure explains what ATP binding itself does to SERCA and how nonproductive phosphorylation is prevented in E2.

scholarly journals The Structure of the Human Respiratory Syncytial Virus M2-1 Protein Bound to the Interaction Domain of the Phosphoprotein P Defines the Orientation of the Complex

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Muniyandi Selvaraj ◽  
Kavestri Yegambaram ◽  
Eleanor J. A. A. Todd ◽  
Charles-Adrien Richard ◽  
Rachel L. Dods ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Respiratory Syncytial Virus ◽  
Binding Sites ◽  
Rna Virus ◽  
Respiratory Illness ◽  
Human Respiratory Syncytial Virus ◽  
Interaction Domain ◽  
High Affinity ◽  
Hydrogen Bond Interactions ◽  
Syncytial Virus

ABSTRACTHuman respiratory syncytial virus (HRSV) is a negative-stranded RNA virus that causes a globally prevalent respiratory infection, which can cause life-threatening illness, particularly in the young, elderly, and immunocompromised. HRSV multiplication depends on replication and transcription of the HRSV genes by the virus-encoded RNA-dependent RNA polymerase (RdRp). For replication, this complex comprises the phosphoprotein (P) and the large protein (L), whereas for transcription, the M2-1 protein is also required. M2-1 is recruited to the RdRp by interaction with P and also interacts with RNA at overlapping binding sites on the M2-1 surface, such that binding of these partners is mutually exclusive. The molecular basis for the transcriptional requirement of M2-1 is unclear, as is the consequence of competition between P and RNA for M2-1 binding, which is likely a critical step in the transcription mechanism. Here, we report the crystal structure at 2.4 Å of M2-1 bound to the P interaction domain, which comprises P residues 90 to 110. The P90–110 peptide is alpha helical, and its position on the surface of M2-1 defines the orientation of the three transcriptase components within the complex. The M2-1/P interface includes ionic, hydrophobic, and hydrogen bond interactions, and the critical contribution of these contacts to complex formation was assessed using a minigenome assay. The affinity of M2-1 for RNA and P ligands was quantified using fluorescence anisotropy, which showed high-affinity RNAs could outcompete P. This has important implications for the mechanism of transcription, particularly the events surrounding transcription termination and synthesis of poly(A) sequences.IMPORTANCEHuman respiratory syncytial virus (HRSV) is a leading cause of respiratory illness, particularly in the young, elderly, and immunocompromised, and has also been linked to the development of asthma. HRSV replication depends on P and L, whereas transcription also requires M2-1. M2-1 interacts with P and RNA at overlapping binding sites; while these interactions are necessary for transcriptional activity, the mechanism of M2-1 action is unclear. To better understand HRSV transcription, we solved the crystal structure of M2-1 in complex with the minimal P interaction domain, revealing molecular details of the M2-1/P interface and defining the orientation of M2-1 within the tripartite complex. The M2-1/P interaction is relatively weak, suggesting high-affinity RNAs may displace M2-1 from the complex, providing the basis for a new model describing the role of M2-1 in transcription. Recently, the small molecules quercetin and cyclopamine have been used to validate M2-1 as a drug target.

Crystal Structure of the Bovine Lactadherin C2 Domain, a Potential Anticoagulant, Shows Similarity to Factor V and Factor VIII.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 194-194
Author(s):  
Lin Lin ◽  
Qing Huai ◽  
Mingdong Huang ◽  
Bruce Furie ◽  
Barbara C. Furie
Keyword(s):  
Crystal Structure ◽  
Factor Viii ◽  
Blood Coagulation ◽  
Binding Sites ◽  
Coagulation Factors ◽  
Factor V ◽  
C2 Domain ◽  
C2 Domains ◽  
Hydrophobic Residues ◽  
High Affinity

Abstract Lactadherin is a glycoprotein expressed by mammary epithelial cells as a cell surface protein and as an abundantly secreted protein during lactation. Lactadherin is comprised of two EGF-like domains and two C-like domains that share homology with the C domains of blood clotting proteins Factor V (FV) and Factor VIII (FVIII). Similar to these coagulation factors, lactadherin binds to phosphatidylserine (PS) containing phospholipid (PL) membranes with high affinity although lactadherin binding requires lower concentrations of PS and is independent of phosphatidylethanolamine. Lactadherin shows efficient competition for membrane binding sites recognized by vitamin K-dependent coagulation factors as well as FV and FVIII with half-maximal displacement of these proteins from PS containing PL vesicles at lactadherin concentrations of 1–4 nM. Lactadherin inhibits the tenase complex, the prothrombinase complex, and the factor VIIa-tissue factor complex. Thus, it has been suggested as an anticoagulant through competition for PL binding sites with blood coagulation proteins. As blood coagulation factors VIII and V bind to PL membranes via C domains with high affinity and specificity, we have determined the crystal structure of the C2 domain of bovine lactadherin (residues 1 to 158) for comparison with the crystal structures of the C2 domains of FV and FVIII. The lactadherin C2 domain was cloned from a bovine EST clone, over-expressed in Pichia pastoris, purified by ion-exchange and gel filtration chromatography and crystallized by the vapor diffusion method. The crystals diffracted to 2.4 Å and have cell dimension of a=108.12Å, b=107.79Å, c= 82.75Å and belong to space group P212121. The structure was determined and refined at a resolution of 2.4 Å. There are four molecules per asymmetry unit. The overall structure of the lactadherin C2 is similar to the C2 domains of FV and FVIII (root-mean-square-deviation of Cα atoms of 0.9 Å and 1.2 Å, respectively). Similarly to the FV (Macedo-Ribeiro, S et al, Nature 1999, 402:434–439) and FVIII (Pratt, KP et al, Nature, 1999, 402:439–441) C2 domains, the lactadherin C2 domain consists of eight major antiparallel strands arranged in two β-sheets of five and three strands packed against one another with the N- and C-terminal regions linked by a disulfide bridge. Like the FV and FVIII C2 domains, the lactadherin C2 domain structure reveals a β-sheet-sandwiched core, from which two β-turns and a loop display a group of solvent-exposed hydrophobic residues. The crystal structures suggest that PL binding is mediated by hydrophobic residues in the C2 domains of FV and FVIII and multiple mutagenesis and antibody inhibition studies support this hypothesis. Based on the crystal structure it is likely that residues Trp26, Phe31, Thr53, Phe81, and Gly82 of lactadherin participate in PL binding. The conformations of regions involved in PL binding are quite different from that of FV and FVIII. The C2 domain of lactadherin may thus have the potential to serve as a unique anticoagulant for treatment of thrombotic disease by blocking the action of FV and FVIII.

Engineering a High-Affinity Anti-IL-15 Antibody: Crystal Structure Reveals an α-Helix in VH CDR3 as Key Component of Paratope

2011 ◽  
Vol 406 (1) ◽  
pp. 160-175 ◽  
Author(s):  
David C. Lowe ◽  
Stefan Gerhardt ◽  
Alison Ward ◽  
David Hargreaves ◽  
Malcolm Anderson ◽  
...  
Keyword(s):  
Crystal Structure ◽  
High Affinity ◽  
Α Helix

Structure of oxalate-substituted diferric mare lactoferrin at 2.7 Å resolution

1999 ◽  
Vol 55 (11) ◽  
pp. 1792-1798 ◽  
Author(s):  
Ashwani K. Sharma ◽  
Tej P. Singh
Keyword(s):  
Crystal Structure ◽  
Metal Ions ◽  
Metal Binding ◽  
Binding Sites ◽  
Metal Ion ◽  
Anion Binding ◽  
Human Lactoferrin ◽  
Final Model ◽  
High Affinity ◽  
Oxalate Anion

Lactoferrin binds two Fe3+ and two CO^{2-}_{3} ions with high affinity. It can also bind other metal ions and anions. In order to determine the perturbations in the environments of the binding sites in the N and C lobes and elsewhere in the protein, the crystal structure of oxalate-substituted diferric mare lactoferrin has been determined at 2.7 Å resolution. The final model has a crystallographic R factor of 21.3% for all data in the resolution range 17.0–2.7 Å. The substitution of an oxalate anion does not perturb the overall structure of the protein, but produces several significant changes at the metal-binding and anion-binding sites. The binding of the oxalate anion is symmetrical in both the N and C lobes, unlike in diferric dioxalate human lactoferrin, where the oxalate anion binds the metal ion symmetrically in the C lobe and asymmetrically in the N lobe.

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