The solution structures of mutant calbindin D9k's, as determined by NMR, show that the calcium binding site can adopt different folds

Johansson, Charlotta; Ullner, Magnus; Drakenberg, Torbjoern

doi:10.1021/bi00084a007

Cited by 25 publications

(27 citation statements)

References 45 publications

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“…A comparison of the CD spectra, stability toward guanidine-HCl, and the quaternary structure of wtS100A2 and the N mutant suggest that the mutation did not dramatically affect the global folding, although local conformational changes occur (see Tyr fluorescence), probably in the region involved in hydrophobic interactions (see bis-ANS fluorescence). A very similar loop grafting experiment on calbindin D 9k also confirmed that the overall structure remains the same (50). But the N-terminal segment (4 -6 residues) in the loop of site I, which is normally turned "inside out" and coordinates Ca 2ϩ by main chain carbonyls, is in the mutant reoriented "outside out" as in canonical EFhands and coordinates Ca 2ϩ with the side chain carboxylates.…”

Section: Ca 2ϩ and Zn 2ϩ Binding To S100a2 And Mutants 18831supporting

confidence: 53%

Binding of Ca2+ and Zn2+ to Human Nuclear S100A2 and Mutant Proteins

Franz¹,

Durussel²,

Cox³

et al. 1998

Journal of Biological Chemistry

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Section: Ca 2ϩ and Zn 2ϩ Binding To S100a2 And Mutants 18831supporting

confidence: 53%

Binding of Ca2+ and Zn2+ to Human Nuclear S100A2 and Mutant Proteins

Franz¹,

Durussel²,

Cox³

et al. 1998

Journal of Biological Chemistry

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“…Many of the 33 residues have NH and C"H shifts that are very similar to the shifts in intact calciumloaded calbindin D9k. A majority of the NH-C"H crosspeaks in the COSY spectrum could thus be identified by comparison with the-shifts in P43M (Johansson et al, 1993). These preliminary assignments were then confirmed by NH-NH and NH-C" H crosspeaks in the 200ms NOESY spectrum, which also led to identification of all NH and/or C"H resonances except for S44, T45, E5 1, and E52.…”

Section: ' H Nmr Assignments Of (Cysf2) Camentioning

confidence: 74%

“…D58 and K72 have C"H shifts close to the water resonance at 37 "C and the C"H shifts are therefore set to 4.65. Structural shifts (i.e., chemical shifts minus random coil shifts [Wuthrich, 19861) of backbone protons in the Caz form of (L39C + P43M + 173C) are shown in Figure 9 in comparison with P43M (Johansson et al, 1993). On comparing the two proteins it appears that the disulfide bridge is well accommodated and the only region where minor conformational changes cannot be excluded is in the close vicinity of the SS-bond itself.…”

Section: 'H Nmr Assignments Of (L39c + P43m + I73c)mentioning

confidence: 99%

Disulfide bonds in homo‐ and heterodimers of EF‐hand subdomains of calbindin D_9k: Stability, calcium binding, and NMR studies

Linse¹,

Thulin²,

Sellers³

1993

Protein Science

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The effect of decreased protein flexibility on the stability and calcium binding properties of calbindin D9k has been addressed in studies of a disulfide bridged calbindin Dgk mutant, denoted (L39C + P43M + I73C), with substitutions Leu 39 -+ Cys, Ile 73 -+ Cys, and Pro 43 + Met. Backbone 'H NMR assignments show that the disulfide bond, which forms spontaneously under air oxidation, is well accommodated. The disulfide is inserted on the opposite end of the protein molecule with respect to the calcium sites, to avoid direct interference with these sites, as confirmed by Ii3Cd NMR. The effect of the disulfide bond on calcium binding was assessed by titrations in the presence of a chromophoric chelator. A small but significant effect on the cooperativity was found, as well as a very modest reduction in calcium affinity. The disulfide bond increases T,,,, the transition midpoint of thermal denaturation, of calcium free calbindin D9k from 85 to 95 "C and C,,,, the urea concentration of half denaturation, from 5.3 to 8.0 M. Calbindins with one covalent bond linking the two EF-hand subdomains are equally stable regardless if the covalent link is the 43-44 peptide bond or the disulfide bond.Kinetic remixing experiments show that separated CNBr fragments of (L39C + P43M + I73C), each comprising one EF-hand, form disulfide linked homodimers. Each homodimer binds two calcium ions with positive cooperativity, and an average affinity of IO6 M-I. Disulfide linkage dramatically increases the stability of each homodimer. For the homodimer of the C-terminal fragment T,,, increases from 59 k 2 without covalent linkage to 91 k 2 "C with disulfide, and C, from = 1.5 to 7.5 M. The overall topology of this homodimer is derived from 'H NMR assignments and a few key NOES.

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“…As described below, NMR hydrogen exchange data indicate a subtler effect of the mutation, that involves the uncoupling of the dynamics of subdomain 1 from 2. The P405M-HlyIIC mutant was selected because a bulky methionine should most favor a trans peptide bond 33 . However, P405A-HlyIIC with a smaller alanine substitution, has a similar 1 H- 15 N HSQC spectrum indicating a conserved structure, similar circular dichroism (CD) spectra, and a similar stability to thermal unfolding within 1 degree for that of P405M-HlyIIC (Fig.…”

Section: Resultsmentioning

confidence: 99%

NMR structure of the Bacillus cereus hemolysin II C-terminal domain reveals a novel fold

Kaplan

Kaus

et al. 2017

Sci Rep

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In addition to multiple virulence factors, Bacillus cereus a pathogen that causes food poisoning and life-threatening wound infections, secretes the pore-forming toxin hemolysin II (HlyII). The HlyII toxin has a unique 94 amino acid C-terminal domain (HlyIIC). HlyIIC exhibits splitting of NMR resonances due to cis/trans isomerization of a single proline near the C-terminus. To overcome heterogeneity, we solved the structure of P405M-HlyIIC, a mutant that exclusively stabilizes the trans state. The NMR structure of HlyIIC reveals a novel fold, consisting of two subdomains αA-β1-β2 and β3-β4-αB-β5, that come together in a barrel-like structure. The barrel core is fastened by three layers of hydrophobic residues. The barrel end opposite the HlyIIC-core has a positively charged surface, that by binding negatively charged moieties on cellular membranes, may play a role in target-cell surface recognition or stabilization of the heptameric pore complex. In the WT domain, dynamic flexibility occurs at the N-terminus and the first α-helix that connects the HlyIIC domain to the HlyII-core structure. In the destabilizing P405M mutant, increased flexibility is evident throughout the first subdomain, suggesting that the HlyIIC structure may have arisen through gene fusion.The soil-dwelling, spore-forming B. cereus bacterium 1-3 produces a number of virulence factors 4 including several secreted pore-forming toxins (PFTs) that form lytic channels in the membranes of target cells 5 . One of these toxins, hemolysin II (HlyII), is present in several closely related Bacillus species including B. cereus, B. thuringiensis (a bacterium that parasitizes insects and has insecticide applications), and B. anthracis (the cause of anthrax) 6, 7 . In B. cereus, expression of HlyII is under the control of the HlyIIR protein 8 , and the Fur system that regulates iron homeostasis 9,10 . Expression of HlyII is greater under oxic conditions than under conditions mimicking the intestinal tract, suggesting the toxin may not play a major role in gastrointestinal disease 11 . Although the physiological target of HlyII is not known, the purified toxin lyses rabbit and human erythrocytes 12 as well as other cultured mammalian cells 13,14 . In addition, the toxin can attack species like algae 15 and insects. Studies in mice and insects suggest that HlyII is involved in virulence, and that the toxin causes apoptosis of macrophages in vitro and in vivo 16 . HlyII belongs to a larger family of secreted toxins with similar predicted core structures including the B. cereus cytotoxin K (CytK) 17 , Staphylococcal hemolysins/leukocidins 18,19 , and toxins secreted by a variety of Vibrio species 20 . Similar to other family members, HlyII is secreted as a water-soluble monomer that assembles into a heptameric pore following binding to cell membranes 12,21 . A unique feature of HlyII is the attachment of a C-terminal domain consisting of 94 amino acids that shows no sequence or structural homology to other known proteins 18 . The C-terminal domain, henceforth r...

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The solution structures of mutant calbindin D9k's, as determined by NMR, show that the calcium binding site can adopt different folds

Cited by 25 publications

References 45 publications

Binding of Ca2+ and Zn2+ to Human Nuclear S100A2 and Mutant Proteins

Binding of Ca2+ and Zn2+ to Human Nuclear S100A2 and Mutant Proteins

Disulfide bonds in homo‐ and heterodimers of EF‐hand subdomains of calbindin D_9k: Stability, calcium binding, and NMR studies

NMR structure of the Bacillus cereus hemolysin II C-terminal domain reveals a novel fold

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The solution structures of mutant calbindin D9k's, as determined by NMR, show that the calcium binding site can adopt different folds

Cited by 25 publications

References 45 publications

Binding of Ca2+ and Zn2+ to Human Nuclear S100A2 and Mutant Proteins

Binding of Ca2+ and Zn2+ to Human Nuclear S100A2 and Mutant Proteins

Disulfide bonds in homo‐ and heterodimers of EF‐hand subdomains of calbindin D9k: Stability, calcium binding, and NMR studies

NMR structure of the Bacillus cereus hemolysin II C-terminal domain reveals a novel fold

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Disulfide bonds in homo‐ and heterodimers of EF‐hand subdomains of calbindin D_9k: Stability, calcium binding, and NMR studies