Structure of a highly acidic β-lactamase from the moderate halophile<i>Chromohalobacter</i>sp. 560 and the discovery of a Cs<sup>+</sup>-selective binding site

Arai, Shigeki; Yonezawa, Yasushi; Okazaki, Nobuo; Matsumoto, Fumiko; Shibazaki, C.; Shimizu, R; Yamada, Michihiro; Adachi, Motoyasu; Tamada, Taro; Kawamoto, Masahide; Tokunaga, Hiroko; Ishibashi, Matsujiro; Blaber, Michael; Tokunaga, Masao; Kuroki, Ryota

doi:10.1107/s1399004714027734

Cited by 11 publications

(7 citation statements)

References 49 publications

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“…Comparison with experimental data suggests that under non-natural electrolyte conditions, the native fold of some of the proteins studied here indeed continues to be the stable state. This seems to be the case for the halophilic and mesophilic carbonic anhydrases ( 12 ) and the halophilic β -lactamase ( 69 ) studied here: experimental measurements of protein activity indicate that these proteins remain active–and thus presumably folded and in soluble form–under non-natural KCl concentrations. For other proteins, however, the conformational stability seen in the simulation may reflect a metastable state with lifetime longer than the simulation time.…”

Section: Resultsmentioning

confidence: 66%

Proteins maintain hydration at high [KCl] concentration regardless of content in acidic amino acids

Daronkola

Verde

2021

Biophysical Journal

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Proteins of halophilic organisms, which accumulate molar concentrations of KCl in their cytoplasm, have a much higher content in acidic amino acids than proteins of mesophilic organisms. It has been proposed that this excess is necessary to maintain proteins hydrated in an environment with low water activity, either via direct interactions between water and the carboxylate groups of acidic amino acids or via cooperative interactions between acidic amino acids and hydrated cations. Our simulation study of five halophilic proteins and five mesophilic counterparts does not support either possibility. The simulations use the AMBER ff14SB force field with newly optimized Lennard-Jones parameters for the interactions between carboxylate groups and potassium ions. We find that proteins with a larger fraction of acidic amino acids indeed have higher hydration levels, as measured by the concentration of water in their hydration shell and the number of water/protein hydrogen bonds. However, the hydration level of each protein is identical at low (b KCl ¼ 0.15 mol/kg) and high (b KCl ¼ 2 mol/kg) KCl concentrations; excess acidic amino acids are clearly not necessary to maintain proteins hydrated at high salt concentration. It has also been proposed that cooperative interactions between acidic amino acids in halophilic proteins and hydrated cations stabilize the folded protein structure and would lead to slower dynamics of the solvation shell. We find that the translational dynamics of the solvation shell is barely distinguishable between halophilic and mesophilic proteins; if such a cooperative effect exists, it does not have that entropic signature.

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Section: Resultsmentioning

confidence: 66%

Proteins maintain hydration at high [KCl] concentration regardless of content in acidic amino acids

Daronkola

Verde

2021

Biophysical Journal

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“…This tight binding of hydration water has previously been observed in crystal structures of halophilic proteins. [23][24][25] However it is not certain whether this was a true feature or an artefact of crystallisation. The solvation-stabilisation theory 4 states this tight binding as the possible mechanism of the stability of halophilic proteins, even at molar salt concentrations.…”

Section: Discussionmentioning

confidence: 99%

“…19,22 However, while the binding of ions to the negatively charged surface has been observed in crystallographic structures of halophilic proteins, other studies have failed to show large numbers of ions. [23][24][25] This discrepancy could be due to the requirement of specific additives in the crystallogenesis process aimed at crystal growth rather than representing in vivo conditions. Solvent molecules present on the surface may be an artefact of the crystallisation process; therefore caution has to be taken when making an inference from this type of data.…”

Section: Introductionmentioning

confidence: 99%

Structural evidence for solvent-stabilisation by aspartic acid as a mechanism for halophilic protein stability in high salt concentrations

Lenton

Walsh

Rhys

et al. 2016

Phys. Chem. Chem. Phys.

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Halophilic organisms have adapted to survive in high salt environments, where mesophilic organisms would perish. One of the biggest 4 challenges faced by halophilic proteins is the ability to maintain both structure and function under molar concentrations of salt. A distinct 5 adaptation of halophilic proteins, compared to mesophilic homologues, is the abundance of aspartic acid on the protein surface. 6Mutagenesis and crystallographic studies of halophilic proteins suggest an important role for solvent interactions with the surface aspartic 7 acid residues. This interaction, between the regions of acidic protein surface and solvent, is thought to maintain a hydration layer around 8 the protein under molar salt concentrations thereby allowing halophilic proteins to retain their functional state. Here we present neutron 9 diffraction data of monomeric zwitterionic form of aspartic acid solutions at physiological pH in 0.25 M and 2.5 M concentration of 10 potassium chloride, to mimic mesophilic and halophilic-like environmental conditions. We have used isotopic substitution in combination 11 with empirical potential structure refinement to extract atomic-scale information from the data. Our study provides structural insight that 12 support the hypothesis that carboxyl groups on acidic residues bind water more tightly under high salt conditions, in support of the 13 residue-ion interaction model of halophilic protein stabilisation. Furthermore our data show that in the presence of high salt the self 14 association between the zwitterionic form of aspartic acid molecules is reduced, suggesting a possible mechanism through which protein 15 aggregation is prevented.

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“…560, PDB 3ws4) where equal concentrations of Sr 2+ and Ca 2+ (200 mM each) were used for crystallization. 40 Three Ca 2+ -sites (referred to as sites 2, 6, and 7) were found to be occupied by Sr 2+ , which retained the Ca 2+ protein ligands except in site 2, where two Asp bidentately bound to Ca 2+ were replaced by a different Asp upon binding Sr 2+ . At sites 2 or 6 in PDB 3ws4, Sr 2+ is bound by two Asp − monodentately, whereas at site 7, Sr 2+ is bound by Glu352 bidentately (Figure 2).…”

Section: ■ Discussionmentioning

confidence: 99%

Protein Ca²⁺-Sites Prone to Sr²⁺ Substitution: Implications for Strontium Therapy

et al. 2023

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Strontium (Sr), an alkali metal with properties similar to calcium, in the form of soluble salts is used to treat osteoporosis. Despite the information accumulated on the role of Sr2+ as a Ca2+ mimetic in biology and medicine, there is no systematic study of how the outcome of the competition between the two dications depends on the physicochemical properties of (i) the metal ions, (ii) the first- and second-shell ligands, and (iii) the protein matrix. Specifically, the key features of a Ca2+-binding protein that enable Sr2+ to displace Ca2+ remain unclear. To address this, we studied the competition between Ca2+ and Sr2+ in protein Ca2+-binding sites using density functional theory combined with the polarizable continuum model. Our findings indicate that Ca2+-sites with multiple strong charge-donating protein ligands, including one or more bidentately bound Asp–/Glu– that are relatively buried and rigid are protected against Sr2+ attack. On the other hand, Ca2+-sites crowded with multiple protein ligands may be prone to Sr2+ displacement if they are solvent-exposed and flexible enough so that an extra backbone ligand from the outer shell can bind to Sr2+. In addition, solvent-exposed Ca2+ sites with only a few weak charge-donating ligands that can rearrange to fit the strontium’s coordination requirements are susceptible to Sr2+ displacement. We provide the physical basis of these results and discuss potential novel protein targets of therapeutic Sr2+.

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Structure of a highly acidic β-lactamase from the moderate halophileChromohalobactersp. 560 and the discovery of a Cs⁺-selective binding site

Abstract: The tertiary structure of a β-lactamase derived from the halobacterium Chromohalobacter sp. 560 (HaBLA) was determined by X-ray crystallography. Three unique Sr2+-binding sites and one Cs+-binding site were discovered in the HaBLA molecule.

Cited by 11 publications

References 49 publications

Proteins maintain hydration at high [KCl] concentration regardless of content in acidic amino acids

Proteins maintain hydration at high [KCl] concentration regardless of content in acidic amino acids

Structural evidence for solvent-stabilisation by aspartic acid as a mechanism for halophilic protein stability in high salt concentrations

Protein Ca²⁺-Sites Prone to Sr²⁺ Substitution: Implications for Strontium Therapy

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Structure of a highly acidic β-lactamase from the moderate halophileChromohalobactersp. 560 and the discovery of a Cs+-selective binding site

Abstract: The tertiary structure of a β-lactamase derived from the halobacterium Chromohalobacter sp. 560 (HaBLA) was determined by X-ray crystallography. Three unique Sr2+-binding sites and one Cs+-binding site were discovered in the HaBLA molecule.

Cited by 11 publications

References 49 publications

Proteins maintain hydration at high [KCl] concentration regardless of content in acidic amino acids

Proteins maintain hydration at high [KCl] concentration regardless of content in acidic amino acids

Structural evidence for solvent-stabilisation by aspartic acid as a mechanism for halophilic protein stability in high salt concentrations

Protein Ca2+-Sites Prone to Sr2+ Substitution: Implications for Strontium Therapy

Contact Info

Product

Resources

About

Structure of a highly acidic β-lactamase from the moderate halophileChromohalobactersp. 560 and the discovery of a Cs⁺-selective binding site

Protein Ca²⁺-Sites Prone to Sr²⁺ Substitution: Implications for Strontium Therapy