Zn Binding Sites Research Articles

Divalent cations influence the dimerization mode of murine S100A9 protein by modulating its disulfide bond pattern

S100A9, with its congener S100A8, belongs to the S100 family of calcium-binding proteins found exclusively in vertebrates. These two proteins are major constituents of neutrophils. In response to a pathological condition, they can be released extracellularly and become alarmins that induce both pro- and anti-inflammatory signals, through specific cell surface receptors. They also act as antimicrobial agents, mainly as a S100A8/A9 heterocomplex, through metal sequestration. The mechanisms whereby divalent cations modulate the extracellular functions of S100A8 and S100A9 are still unclear. Importantly, it has been proposed that these ions may affect both the ternary and quaternary structure of these proteins, thereby influencing their physiological properties. In the present study, we report the crystal structures of WT and C80A murine S100A9 (mS100A9), determined at 1.45 and 2.35 Å resolution, respectively, in the presence of calcium and zinc. These structures reveal a canonical homodimeric form for the protein. They also unravel an intramolecular disulfide bridge that stabilizes the C-terminal tail in a rigid conformation, thus shaping a second Zn-binding site per S100A9 protomer. In solution, mS100A9 apparently binds only two zinc ions per homodimer, with an affinity in the micromolar range, and aggregates in the presence of excess zinc. Using mass spectrometry, we demonstrate that mS100A9 can form both non-covalent and covalent homodimers with distinct disulfide bond patterns. Interestingly, calcium and zinc seem to affect differentially the relative proportion of these forms. We discuss how the metal-dependent interconversion between mS100A9 homodimers may explain the versatility of physiological functions attributed to the protein.

Zinc and SARS‑CoV‑2: A molecular modelingstudy of Zn interactions with RNA‑dependentRNA‑polymerase and 3C‑like proteinase enzymes.

RNA-dependent RNA-polymerase (RdRp) and 3C-like proteinase (3CLpro) are two main enzymes that play a key role in the replication of SARS-CoV-2. Zinc (Zn) has strong immunogenic properties and is known to bind to a number of proteins, modulating their activities. Zn also has a history of use in viral infection control. Thus, the present study models potential Zn binding to RdRp and the 3CLpro. Through molecular modeling, the Zn binding sites in the aforementioned two important enzymes of viral replication were found to be conserved between severe acute respiratory syndrome (SARS)-coronavirus (CoV) and SARS-CoV-2. The location of these sites may influence the enzymatic activity of 3CLpro and RdRp in coronavirus disease 2019 (COVID-19). Since Zn has established immune health benefits, is readily available, non-expensive and a safe food supplement, with the comparisons presented here between SARS-CoV and COVID-19, the present study proposes that Zn could help ameliorate the disease process of COVID-19 infection.

Abstract 12928: Vascular Complications in Covid-19 and Role of Zinc in Viral Inhibition

Although COVID-19 is associated with severe respiratory dysfunctions, conspicuous vascular complications and neurological manifestations have been reported worldwide. Of note, two distinctive features have been noticed in severe patients, progressive increase of inflammation and an unusual trend of hypercoagulation. Interestingly, evidence is mounting that healthy blood vessels protect children from serious effects of COVID-19, such as stroke. These findings suggest vascular complications play a key role in the progress of COVID-19, warranting an investigation to its pathophysiology and treatment strategy related to vascular cells. Cell entry of this SARS-CoV-2 virus depends on binding of the viral spike (S) proteins to cellular receptor ACE2, which could be a key target for blocking the viral entry into host cells. ACE2 is a zinc (Zn) binding metallopeptidase while Zn possesses distinct antiviral properties against many human viruses including coronaviruses. Although the mechanistic studies are lacking, Zn appears to inhibit viral protease and polymerase enzymatic processes, and physical processes such as virus attachment, cell entry, and uncoating. In fact, our data showed that ACE2 has multiple affinity binding sites for Zn. Excess bindings of ionic Zn to ACE2 led to its conformational or functional change, therefore, interfering with its ability to metabolize its substrate as well as inhibiting its binding to S protein. Computational modeling also revealed that one critical Zn binding motif is located in ACE2’s binding domain to S protein, and docking affinity of S protein to ACE2 was significantly reduced after Zn binding to this specific site. Moreover, cell and animal studies using pseudo-virus bearing CoV-2-S protein validated that significantly lower infection of vascular cells in the presence of Zn was observed. Thus, targeting vascular complications in COVID-19 may offer strong benefits including the potential therapeutic role of Zn.

XANES Measurements for Studies of Adsorbed Protein Layers at Liquid Interfaces.

X-ray absorption near edge structure (XANES) spectra for protein layers adsorbed at liquid interfaces in a Langmuir trough have been recorded for the first time. We studied the parkin protein (so-called E3 ubiquitin ligase), which plays an important role in pathogenesis of Parkinson disease. Parkin contains eight Zn binding sites, consisting of cysteine and histidine residues in a tetracoordinated geometry. Zn K-edge XANES spectra were collected in the following two series: under mild radiation condition of measurements (short exposition time) and with high X-ray radiation load. XANES fingerprint analysis was applied to obtain information on ligand environments around zinc ions. Two types of zinc coordination geometry were identified depending on X-ray radiation load. We found that, under mild conditions, local zinc environment in our parkin preparations was very similar to that identified in hemoglobin, treated with a solution of ZnCl2 salt. Under high X-ray radiation load, considerable changes in the zinc site structure were observed; local zinc environment appeared to be almost identical to that defined in Zn-containing enzyme alkaline phosphatase. The formation of a similar metal site in unrelated protein molecules, observed in our experiments, highlights the significance of metal binding templates as essential structural modules in protein macromolecules.

A novel FRET peptide assay reveals efficient Helicobacter pylori HtrA inhibition through zinc and copper binding

Helicobacter pylori (H. pylori) secretes the chaperone and serine protease high temperature requirement A (HtrA) that cleaves gastric epithelial cell surface proteins to disrupt the epithelial integrity and barrier function. First inhibitory lead structures have demonstrated the essential role of HtrA in H. pylori physiology and pathogenesis. Comprehensive drug discovery techniques allowing high-throughput screening are now required to develop effective compounds. Here, we designed a novel fluorescence resonance energy transfer (FRET) peptide derived from a gel-based label-free proteomic approach (direct in-gel profiling of protease specificity) as a valuable substrate for H. pylori HtrA. Since serine proteases are often sensitive to metal ions, we investigated the influence of different divalent ions on the activity of HtrA. We identified Zn++ and Cu++ ions as inhibitors of H. pylori HtrA activity, as monitored by in vitro cleavage experiments using casein or E-cadherin as substrates and in the FRET peptide assay. Putative binding sites for Zn++ and Cu++ were then analyzed in thermal shift and microscale thermophoresis assays. The findings of this study will contribute to the development of novel metal ion-dependent protease inhibitors, which might help to fight bacterial infections.

Facile synthesis of 3D hierarchical micro-/nanostructures in capillaries for efficient capture of circulating tumor cells

The efficient capture of rare circulating tumor cells (CTCs) with high viability is of great importance in cancer diagnosis. The integration of three-dimensional (3D) nanobiointerfaces with capillary flow channel platforms can efficiently improve CTC capture performance by providing abundant binding sites and increasing the likelihood of contact as samples flow through the microchannels. However, due to the complex preparation processes, facile synthesis of nanostructures for use as substrates in flow channels for biomedical applications is still challenging. To reduce the encapsulation steps in the fabricating of nanostructured flow channel devices, we chose the enclosed glass capillaries as flow channels and accomplished all the experiments in the microchannels, including 3D nanostructure synthesis, surface modification and capture/release of CTCs. In this work, we constructed a novel 3D Zn(OH)F/ZnO nanoforest array in capillaries for CTC isolation via a facile microfluidic wet chemistry method. Because of the abundant binding sites of the 3D Zn(OH)F/ZnO nanoforest array, the capture efficiency was remarkably enhanced compared with that of vertical nanowires (90.3% vs 69.1%). In addition, a high release efficiency and cell viability of released cells were achieved by grafting poly(N-isopropylacrylamide) (PNIPPAm). These results may provide evidence for a novel method to fabricate hierarchical 3D substrates with a combination of biomolecule recognition and topographical interaction for biomedical applications.

Phytoremediation of soil heavy metals (Cd and Zn) by castor seedlings: Tolerance, accumulation and subcellular distribution

Cd and Zn pollution was observed to often occur simultaneously in soils. However, previous studies focused on single heavy metal instead of Cd and Zn combined pollution. Castor (Ricinus communis) is considered to have great potential for contaminated soil remediation. The resistance of castor seedlings to heavy metals and the mechanism behind it remain unknown. In this study, the tolerance and accumulation ability of castor seedlings to Cd and Zn were investigated, and the accumulation mechanism involving the subcellular distribution in different tissues was further explored. The results on biomass and chlorophyll revealed that castor seedlings have good tolerance to the pollution with 0–5 mg/kg Cd and 380 mg/kg Zn, while not to the heavy pollution with 25 mg/kg Cd and 380 mg/kg Zn. The maximum accumulation concentrations of Cd and Zn, 175.3 mg Cd/kg and 386.8 mg/kg Zn, appeared in castor seedling root instead of stem and leaf, indicating that root played a significant part in accumulating Zn and Cd. The relative low dosage of Cd (0–5 mg/kg) promoted the accumulation of Zn in the subcellular component, while high dosage (25 mg/kg) inhibited the accumulation of Zn. In subcellular accumulation and distribution of castor seedlings, Cd (27.1%–69.4%) and Zn (39.6%–66.6%) in the cell wall was the highest. With the increase of Cd addition, the accumulation of Cd increased in cell wall while decreased in organelle and soluble fraction. Hydroxyl, amino, amides and carboxyl functional groups on cell wall might provided the main binding sites for Cd and Zn.

Enhancement of Oxidative Reaction by the Intramolecular Electron Transfer between the Coordinated Redox-Active Metal Ions in SOD1

The denatured Cu, Zn superoxide dismutase (SOD1) has the pro-oxidant activity that is suggested to be related with the pathogenesis of amyotrophic lateral sclerosis (ALS). We showed from the changes in the coordinated metal ions that the Cu ion in the Cu-binding site is the catalytic site of the pro-oxidant activity, and a redox-active metal ion in the Zn-binding site has the auxiliary function to enhance the pro-oxidant activity. The auxiliary function is suggested to arise from the intramolecular electron transfer between the coordinated metal ions in the denatured SOD1. The oxidation/reduction cycle of Cu in the Cu-binding site is assisted with changing the oxidation state of a metal ion in the Zn-binding site. The magnitude of the toxicity of the denatured SOD1 is discussed based on the ability of the auxiliary function.

Factors Governing the Different Functions of Zn2+-Sites with Identical Ligands in Proteins.

In Zn-proteins, structural Zn-sites are mostly Cys-rich lined by two or more Cys residues, whereas catalytic Zn-sites usually contain His or Asp/Glu residues and a water molecule. Here, we reveal many examples outside this trend with Zn2+ bound to ligands commonly found in both structural and catalytic Zn-sites, namely, Zn-CC(C/H)x (x = D, E, or H2O) sites. We show that these atypical Zn-sites are found in all known life forms (i.e., eukaryotes, bacteria, archaea, and viruses) and can serve structural roles in some proteins but catalytic roles in others. By calculating the physical properties of these atypical Zn-binding sites, we elucidate why Zn-CC(C/H)x sites of the same composition can serve structural and catalytic roles in proteins. Furthermore, we found new sequence/structural motifs characteristic of catalytic Zn-CCHw sites and provide guidelines to predict the structural/catalytic role of atypical Zn-CC(C/H)x sites of unknown function. We discuss how our results could help to design inhibitors targeting catalytic Zn-CC(C/H) H2O sites.

Molecular characterization and expression analysis of copper-zinc superoxide dismutases from the freshwater alga Closterium ehrenbergii under metal stress.

Superoxide dismutase (SOD) acts as the first line of defense against reactive oxygen species (ROS) within cells. In the present study, we determined two novel CuZnSOD genes (designated as CeCSD1 and CeCSD2) from the toxicity-testing freshwater algae Closterium ehrenbergii and examined their structural features, phylogenetic relationships, and gene expression under exposure to different metals. Putative CeCSD1 (204 aa, 20.6 kDa) and CeCSD2 (155 aa, 15.3 kDa) proteins had conserved CuZnSOD family motifs and metal (Cu, Zn) binding sites, but different N-terminus structures, that is, CeCSD1 has a signal peptide to chloroplasts. Phylogenetic analysis of each protein revealed that C. ehrenbergii was well clustered with other green algae and plants. Real-time PCR results showed that the gene expression obviously increased with heavy metal exposure. In addition, excess copper considerably increased the SOD activity and ROS generation but decreased the photosynthetic efficiency in treated cells. These results suggest that CeCSDs are involved in the antioxidant defense system and can be regarded as potential biomarkers for monitoring metal contaminants in aquatic environments.

Isolation and identification of zinc-chelating peptides from sea cucumber (Stichopus japonicus) protein hydrolysate.

Zinc is known to play an essential role in the biological activities in the human body. In this study, a zinc-chelating peptide (ZCP) produced by Alcalase-assisted hydrolysis of the body wall of sea cucumber was isolated and identified. The ZCP was purified stepwise by ultrafiltration, anion-exchange chromatography, and gel filtration chromatography, in conjunction with ultraviolet-visual (UV-visual) spectrophotometry, which was used to analyze each purified fraction. Analysis of the purified ZCP revealed that its zinc-chelating ability was 33.31%. Analysis of isothermal titration calorimetry suggested that the binding of ZCP and zinc (N ≈ 2) was endothermic, with weak binding affinity. Fourier transform infrared spectroscopy spectra (FTIR) indicated that carboxylic and amide groups in ZCP were the primary binding sites of Zn. Sequencing the result by ultra-performance liquid chromatography-quadrupole/time of flight mass spectrometry (UPLC-Q-TOF-MS/MS) showed that a representative ZCP had the sequence WLTPTYPE with a molecular weight of 1005.5 Da. These results provide a promising foundation for the production of zinc supplements from sea-cucumber-derived ZCPs. © 2019 Society of Chemical Industry.

Toward understanding the binding of Zn in soils by two-dimensional correlation spectroscopy and synchrotron-radiation-based spectromicroscopies

Zinc (Zn) is an essential micronutrient that is generally deficient in soils to accommodate plant needs. Therefore, it is imperative to enhance the concentration and availability of Zn in soils and plants through fertilization. Currently, however, the binding characteristics and fate of Zn in soils under various fertilization regimes remain unclear. Here, two-dimensional correlation spectroscopy (2DCOS) and synchrotron-radiation-based spectromicroscopies were used to identify functional groups responsible for Zn binding in soils following different fertilization treatments. The results showed that a 23-year long-term manure application significantly increased the concentrations of Zn in both soil and corn grain as compared to those having received a traditional inorganic NPK treatment. The 2DCOS analysis of FTIR spectra showed that aliphatic C mainly contributed to Zn binding in the NPK-treated soils, whereas SiO groups played a dominant role for Zn binding in the manure-treated soils. In soil dissolved organic matter, Zn2+ was first bound with FeO, followed by aliphatic C and polysaccharide C in the NPK treatment, but bound with aliphatic CH prior to SiO in the manure treatment. Furthermore, synchrotron-radiation-based FTIR and μ-XRF spectromicroscopies indicated that the distribution of Zn, clay minerals, sesquioxides and C functional groups was heterogeneous at the micro-scale, suggesting a heterogeneous binding site for Zn in soils. Using μ-XRF spectromicroscopies, a strong spatial correlation at the submicron scale between Fe and Zn was found in soils, suggesting that Fe-bearing minerals, likely Fe (hydr) oxides, contributed binding sites for Zn. Together, these results indicate that combining 2DCOS and synchrotron-radiation-based spectromicroscopies yields useful information for exploring the binding mechanisms among micronutrients, minerals and organic components in soils, particularly revealing how binding sites for micronutrients in soils can be modified according to fertilization regime.

Investigation on the metal binding sites of a putative Zn(ii) transporter in opportunistic yeast species Candida albicans

The protein fragment C4YJH2 of Candida albicans has the ability to tightly coordinate Zn(ii) at its C-terminal region by means of an evolutionarily well-conserved histidine-rich sequence.

Binding of Zn(II) to Tropomyosin Receptor Kinase A in Complex with Its Cognate Nerve Growth Factor: Insights from Molecular Simulation and in Vitro Essays.

The binding of the human nerve growth factor (NGF) protein to tropomyosin receptor kinase A (TrkA) is associated with Alzhemeir's development. Owing to the large presence of zinc(II) ions in the synaptic compartments, the zinc ions might be bound to the complex in vivo. Here, we have identified a putative zinc binding site using a combination of computations and experiments. First, we have predicted structural features of the NGF/TrkA complex in an aqueous solution by molecular simulation. Metadynamics free energy calculations suggest that these are very similar to those in the X-ray structure. Here, the "crab" structure of the NGF shape binds tightly to two TrkA "pincers". Transient conformations of the complex include both more extended and more closed conformations. Interestingly, the latter features facial histidines (His60 and His61) among the N-terminal D1-D3 domains, each of which is a potential binding region for biometals. This suggests the presence of a four-His Zn binding site connecting the two chains. To address this issue, we investigated the binding of a D1-D3 domains' peptide mimic by stability constant and nuclear magnetic resonance measurements, complemented by density functional theory-based calculations. Taken together, these establish unambiguously a four-His coordination of the metal ion in the model systems, supporting the presence of our postulated binding site in the NGF/TrkA complex.

Competitive adsorption of Pb(II) and Zn(II) from aqueous solution by modified beer lees in a fixed bed column

Continuous fixed-bed column studies for Pb(II) and Zn(II) simultaneous removal from aqueous solutions were carried out by using the modified beer lees as an absorbent. The effects of weight of adsorbent in each depth of the bed, flow rate and initial concentration were examined. The results show that the adsorption capacity of Pb(II) and Zn(II) both increase with the increase in the influent concentration, and decrease with the increasing of the packing weight of adsorbent and flow rate. Under the optimum conditions with Pb(II) and Zn(II) concentration of 30 mg/L and 15 mg/L, the adsorbent’s packing weight of 4 g, and the flow rate of 1.0 mL/min, the adsorption capacity of Pb(II) and Zn(II) can reach 29.6 mg/g and 5.43 mg/g, respectively. The coefficients of Bed Depth Service Time and Yoon–Nelson models are all above 0.95, which is suitable to describe the continuous adsorption process of Pb(II) and Zn(II) by the modified beer lees and to predict effluent data in column operation under different experimental conditions. The results of EDX confirm that the Pb(II) and Zn(II) ions presence on the surface of the modified beer lees after adsorption process and the FTIR analysis identifies the hydroxyl, aliphatic acids and carboxyl functional groups as the active binding sites for Pb(II) and Zn(II) ions adsorption.

Prior knowledge or freedom of interpretation? A critical look at a recently published classification of "novel" Zn binding sites.

In a recently published article (Yao, Flight, Rouchka, and Moseley, Proteins 2015;83:1470-1487) the authors proposed novel Zn coordination patterns in protein structures, apparently discovered using an unprejudiced approach to the information collected in the Protein data Bank (PDB), which they advocated as superior to the prior-knowledge-informed paradigm. In our assessment of those propositions we demonstrate here that most, if not all, of the "new" coordination geometries are fictitious, as they are based on incorrectly interpreted protein crystal structures, which in themselves are often not error-free. The flaws of interpretation include partial or wrong Zn sites, missed or wrong ligands, ignored crystal symmetry and ligands, etc. In conclusion, we warn against using this and similar meta-analyses that ignore chemical and crystallographic knowledge, and emphasize the importance of safeguarding structural databases against bad apples. Proteins 2016; 84:770-776. © 2016 Wiley Periodicals, Inc.

Zinc and Manganese Chelation by Neutrophil S100A8/A9 (Calprotectin) Limits Extracellular Aspergillus fumigatus Hyphal Growth and Corneal Infection.

Calprotectin, a heterodimer of S100A8 and S100A9, is an abundant neutrophil protein that possesses antimicrobial activity primarily because of its ability to chelate zinc and manganese. In the current study, we showed that neutrophils from calprotectin-deficient S100A9(-/-) mice have an impaired ability to inhibit Aspergillus fumigatus hyphal growth in vitro and in infected corneas in a murine model of fungal keratitis; however, the ability to inhibit hyphal growth was restored in S100A9(-/-) mice by injecting recombinant calprotectin. Furthermore, using recombinant calprotectin with mutations in either the Zn and Mn binding sites or the Mn binding site alone, we show that both zinc and manganese binding are necessary for calprotectin's antihyphal activity. In contrast to hyphae, we found no role for neutrophil calprotectin in uptake or killing of intracellular A. fumigatus conidia either in vitro or in a murine model of pulmonary aspergillosis. We also found that an A. fumigatus ∆zafA mutant, which demonstrates deficient zinc transport, exhibits impaired growth in infected corneas and following incubation with neutrophils or calprotectin in vitro as compared with wild-type. Collectively, these studies demonstrate a novel stage-specific susceptibility of A. fumigatus to zinc and manganese chelation by neutrophil-derived calprotectin.

Dual Action of Zn2+ on the Transport Cycle of the Dopamine Transporter

The dopamine transporter shapes dopaminergic neurotransmission by clearing extracellular dopamine and by replenishing vesicular stores. The dopamine transporter carries an endogenous binding site for Zn(2+), but the nature of the Zn(2+)-dependent modulation has remained elusive: both, inhibition and stimulation of DAT have been reported. Here, we exploited the high time resolution of patch-clamp recordings to examine the effects of Zn(2+) on the transport cycle of DAT: we recorded peak currents associated with substrate translocation and steady-state currents reflecting the forward transport mode of DAT. Zn(2+) depressed the peak current but enhanced the steady-state current through DAT. The parsimonious explanation is preferential binding of Zn(2+) to the outward facing conformation of DAT, which allows for an allosteric activation of DAT, in both, the forward transport mode and substrate exchange mode. We directly confirmed that Zn(2+) dissociated more rapidly from the inward- than from the outward-facing state of DAT. Finally, we formulated a kinetic model for the action of Zn(2+) on DAT that emulated all current experimental observations and accounted for all previous (in part contradictory) findings. Importantly, the model predicts that the intracellular Na(+) concentration determines whether substrate uptake by DAT is stimulated or inhibited by Zn(2+). This prediction was directly verified. The mechanistic framework provided by the current model is of relevance for the rational design of allosteric activators of DAT. These are of interest for treating de novo loss-of-function mutations of DAT associated with neuropsychiatric disorders such as attention deficit hyperactivity disorder (ADHD).

Engineering of a disulfide loop instead of a Zn binding loop restores the anti-proliferative, anti-angiogenic and anti-tumor activities of the N-terminal fragment of endostatin: Mechanistic and therapeutic insights

Although considerable effort has been devoted to understanding the molecular mechanism of endostatin's anti-cancer activity, the role of its Zn bound N-terminal loop has not been completely clarified. To investigate whether Zn binding or the N-terminal loop is involved in the anti-cancer properties of endostatin, we compared the structure and biological activity of a native Zn binding endostatin peptide (ES-Zn) with three variants: a Zn free variant (ES), a variant containing both a Zn binding site and a disulfide bond (ES-SSZn), and a variant including a disulfide loop but incapable of Zn binding (ES-SS). Spectroscopic studies indicated that ES-Zn and ES-SS consist of random coil and β structures, whereas ES-SSZn and ES fold into random coils. Theoretical analysis proposed that ES-Zn and ES-SS have a similar binding site to αVβ3 integrin. The anti-proliferative activity of endostatin was retained by all peptides except ES, and the in vitro anti-angiogenic property was preserved in ES-Zn and ES-SS. Remarkably, breast tumor growth and CD31 activity were inhibited more effectively by ES-SS than by ES-Zn. Therefore, a correlation exists between the N-terminal loop and anti-cancer properties of endostatin fragment and a disulfide loop may be more promising than a Zn binding loop for inhibiting tumor growth.

Structural insights into methanol-stable variants of lipase T6 from Geobacillus stearothermophilus.

Enzymatic production of biodiesel by transesterification of triglycerides and alcohol, catalyzed by lipases, offers an environmentally friendly and efficient alternative to the chemically catalyzed process while using low-grade feedstocks. Methanol is utilized frequently as the alcohol in the reaction due to its reactivity and low cost. However, one of the major drawbacks of the enzymatic system is the presence of high methanol concentrations which leads to methanol-induced unfolding and inactivation of the biocatalyst. Therefore, a methanol-stable lipase is of great interest for the biodiesel industry. In this study, protein engineering was applied to substitute charged surface residues with hydrophobic ones to enhance the stability in methanol of a lipase from Geobacillus stearothermophilus T6. We identified a methanol-stable variant, R374W, and combined it with a variant found previously, H86Y/A269T. The triple mutant, H86Y/A269T/R374W, had a half-life value at 70 % methanol of 324 min which reflects an 87-fold enhanced stability compared to the wild type together with elevated thermostability in buffer and in 50 % methanol. This variant also exhibited an improved biodiesel yield from waste chicken oil compared to commercial Lipolase 100L® and Novozyme® CALB. Crystal structures of the wild type and the methanol-stable variants provided insights regarding structure-stability correlations. The most prominent features were the extensive formation of new hydrogen bonds between surface residues directly or mediated by structural water molecules and the stabilization of Zn and Ca binding sites. Mutation sites were also characterized by lower B-factor values calculated from the X-ray structures indicating improved rigidity.