Α1 Helix Research Articles

Enzymatic characterization of a novel thermostable and alkaline tolerant GH10 xylanase and activity improvement by multiple rational mutagenesis strategies

Thermo-alkaline xylanases are widely applied in paper pulping industry. In this study, a novel thermostable and alkaline tolerant GH10 xylanase (Xyn30Y5) gene from alkaliphilic Bacillus sp. 30Y5 was cloned and the surface-layer homology (SLH) domains truncated enzyme (Xyn30Y5-SLH) was expressed in Escherichia coli. The purified Xyn30Y5-SLH was most active at 70 °C and pH 7.0 and showed the highest specific activity of 349.4 U mg−1. It retained more than 90% activity between pH 6.0 to 9.5 and was stable at pH 6.0–10.0. To improve the activity, 47 mutants were designed based on eight rational strategies and 21 mutants showed higher activity. By combinatorial mutagenesis, the best mutant 3B demonstrated specific activity of 1016.8 U mg−1 with a doubled catalytic efficiency (kcat/Km) and RA601/2h value, accompanied by optimal pH shift to 8.0. The molecular dynamics simulation analysis indicated that the increase of flexibility of α5 helix and loop7 located near to the catalytic residues is likely responsible for its activity improvement. And the decrease of flexibility of the most unstable regions is vital for the thermostablity improvement. This work provided not only a novel thermostable and alkaline tolerant xylanase with industrial application potential but also an effective mutagenesis strategy for xylanase activity improvement.

Locking mixed-lineage kinase domain-like protein in its auto-inhibited state prevents necroptosis.

As an alternative pathway of controlled cell death, necroptosis can be triggered by tumor necrosis factor via the kinases RIPK1/RIPK3 and the effector protein mixed-lineage kinase domain-like protein (MLKL). Upon activation, MLKL oligomerizes and integrates into the plasma membrane via its executioner domain. Here, we present the X-ray and NMR costructures of the human MLKL executioner domain covalently bound via Cys86 to a xanthine class inhibitor. The structures reveal that the compound stabilizes the interaction between the auto-inhibitory brace helix α6 and the four-helix bundle by stacking to Phe148. An NMR-based functional assay observing the conformation of this helix showed that the F148A mutant is unresponsive to the compound, providing further evidence for the importance of this interaction. Real-time and diffusion NMR studies demonstrate that xanthine derivatives inhibit MLKL oligomerization. Finally, we show that the other well-known MLKL inhibitor Necrosulfonamide, which also covalently modifies Cys86, must employ a different mode of action.

Crystal structures of ORFV125 provide insight into orf virus-mediated inhibition of apoptosis.

Premature apoptosis of cells is a strategy utilized by multicellular organisms to counter microbial threats. Orf virus (ORFV) is a large double-stranded DNA virus belonging to the poxviridae. ORFV encodes for an apoptosis inhibitory protein ORFV125 homologous to B-cell lymphoma 2 or Bcl-2 family proteins, which has been shown to inhibit host cell encoded pro-apoptotic Bcl-2 proteins. However, the structural basis of apoptosis inhibition by ORFV125 remains to be clarified. We show that ORFV125 is able to bind to a range of peptides spanning the BH3 motif of human pro-apoptotic Bcl-2 proteins including Bax, Bak, Puma and Hrk with modest to weak affinity. We then determined the crystal structures of ORFV125 alone as well as bound to the highest affinity ligand Bax BH3 motif. ORFV125 adopts a globular Bcl-2 fold comprising 7 α-helices, and utilizes the canonical Bcl-2 binding groove to engage pro-apoptotic host cell Bcl-2 proteins. In contrast with a previously predicted structure, ORFV125 adopts a domain-swapped dimeric topology, where the α1 helix from one protomer is swapped into a neighbouring unit. Furthermore, ORFV125 differs from the conserved architecture of the Bcl-2 binding groove and instead of α3 helix forming one of the binding groove walls, ORFV125 utilizes an extended α2 helix that comprises the equivalent region of helix α3. This results in a subtle variation of previously observed dimeric Bcl-2 architectures in other poxvirus and human encoded Bcl-2 proteins. Overall, our results provide a structural and mechanistic basis for orf virus-mediated inhibition of host cell apoptosis.

Computational Design of 25-mer Peptide Binders of SARS-CoV-2.

SARS-CoV-2 is the novel coronavirus causing the COVID-19 pandemic. To enter human cells, the receptor-binding domain (RBD) of the S1 subunit of SARS-CoV-2 (SARS-CoV-2-RBD) initially binds to the peptidase domain of angiotensin-converting enzyme 2 receptor (ACE2-PD). Using peptides to inhibit SARS-CoV-2-RBD binding to ACE2 is a potential therapeutic solution for COVID-19. A previous study identified a 23-mer peptide (SBP1) that bound to SARS-CoV-2-RBD with comparable KD to ACE2. We employed computational protein design and molecular dynamics (MD) to design SARS-CoV-2-RBD 25-mer peptide binders (SPB25) with better predicted binding affinity than SBP1. Using residues 21–45 of the α1 helix of ACE2-PD as the template, our strategy is employing Rosetta to enhance SPB25 binding affinity to SARS-CoV-2-RBD and avoid disrupting existing favorable interactions by using residues that have not been reported to form favorable interactions with SARS-CoV-2-RBD as designed positions. Designed peptides with better predicted binding affinities, by Rosetta, than SPB25 were subjected to MD validation. The MD results show that five designed peptides (SPB25F8N, SPB25F8R, SPB25L25R, SPB25F8N/L25R, and SPB25F8R/L25R) have better predicted binding affinities, by the MM-GBSA method, than SPB25 and SBP1. This study developed an approach to design SARS-CoV-2-RBD peptide binders, and these peptides may be promising candidates as potential SARS-CoV-2 inhibitors.

Genome-wide functional analysis of hot pepper immune receptors reveals an autonomous NLR clade in seed plants.

Summary Plants possess hundreds of intracellular immune receptors encoding nucleotide‐binding domain leucine‐rich repeat (NLR) proteins. Full‐length NLRs or a specific domain of NLRs often induce plant cell death in the absence of pathogen infection.In this study we used genome‐wide transient expression analysis to identify a group of NLRs (ANLs; ancient and autonomous NLRs) carrying autoactive coiled‐coil (CCA) domains in pepper (Capsicum annuum). CCA‐mediated cell death mimics hypersensitive cell death triggered by the interaction between NLRs and pathogen effectors.Sequence alignment and mutagenesis analyses revealed that the intact α1 helix of CCAs is critical for both CCA‐ and ANL‐mediated cell death. Cell death induced by CCAs does not require NRG1/ADR1 or NRC type helper NLRs, suggesting ANLs may function as singleton NLRs. We also found that CCAs localize to the plasma membrane, as demonstrated for Arabidopsis singleton NLR ZAR1. Extended studies revealed that autoactive CCAs are well conserved in other Solanaceae plants as well as in rice, a monocot plant. Further phylogenetic analyses revealed that ANLs are present in all tested seed plants (spermatophytes).Our study not only uncovers the autonomous NLR clade in plants but also provides powerful resources for dissecting the underlying molecular mechanism of NLR‐mediated cell death in plants.

Phylogenetics-based identification and characterization of a superior 2,3-butanediol dehydrogenase for Zymomonas mobilis expression

BackgroundZymomonas mobilis has recently been shown to be capable of producing the valuable platform biochemical, 2,3-butanediol (2,3-BDO). Despite this capability, the production of high titers of 2,3-BDO is restricted by several physiological parameters. One such bottleneck involves the conversion of acetoin to 2,3-BDO, a step catalyzed by 2,3-butanediol dehydrogenase (Bdh). Several Bdh enzymes have been successfully expressed in Z. mobilis, although a highly active enzyme is yet to be identified for expression in this host. Here, we report the application of a phylogenetic approach to identify and characterize a superior Bdh, followed by validation of its structural attributes using a mutagenesis approach.ResultsOf the 11 distinct bdh genes that were expressed in Z. mobilis, crude extracts expressing Serratia marcescens Bdh (SmBdh) were found to have the highest activity (8.89 µmol/min/mg), when compared to other Bdh enzymes (0.34–2.87 µmol/min/mg). The SmBdh crystal structure was determined through crystallization with cofactor (NAD+) and substrate (acetoin) molecules bound in the active site. Active SmBdh was shown to be a tetramer with the active site populated by a Gln247 residue contributed by the diagonally opposite subunit. SmBdh showed a more extensive supporting hydrogen-bond network in comparison to the other well-studied Bdh enzymes, which enables improved substrate positioning and substrate specificity. This protein also contains a short α6 helix, which provides more efficient entry and exit of molecules from the active site, thereby contributing to enhanced substrate turnover. Extending the α6 helix to mimic the lower activity Enterobacter cloacae (EcBdh) enzyme resulted in reduction of SmBdh function to nearly 3% of the total activity. In great contrast, reduction of the corresponding α6 helix of the EcBdh to mimic the SmBdh structure resulted in ~ 70% increase in its activity.ConclusionsThis study has demonstrated that SmBdh is superior to other Bdhs for expression in Z. mobilis for 2,3-BDO production. SmBdh possesses unique structural features that confer biochemical advantage to this protein. While coordinated active site formation is a unique structural characteristic of this tetrameric complex, the smaller α6 helix and extended hydrogen network contribute towards improved activity and substrate promiscuity of the enzyme.

Rational design of highly potent broad-spectrum enterovirus inhibitors targeting the nonstructural protein 2C.

There is a great need for antiviral drugs to treat enterovirus (EV) and rhinovirus (RV) infections, which can be severe and occasionally life-threatening. The conserved nonstructural protein 2C, which is an AAA+ ATPase, is a promising target for drug development. Here, we present a structure-activity relationship study of a previously identified compound that targets the 2C protein of EV-A71 and several EV-B species members, but not poliovirus (PV) (EV-C species). This compound is structurally related to the Food and Drug Administration (FDA)-approved drug fluoxetine-which also targets 2C-but has favorable chemical properties. We identified several compounds with increased antiviral potency and broadened activity. Four compounds showed broad-spectrum EV and RV activity and inhibited contemporary strains of emerging EVs of public health concern, including EV-A71, coxsackievirus (CV)-A24v, and EV-D68. Importantly, unlike (S)-fluoxetine, these compounds are no longer neuroactive. By raising resistant EV-A71, CV-B3, and EV-D68 variants against one of these inhibitors, we identified novel 2C resistance mutations. Reverse engineering of these mutations revealed a conserved mechanism of resistance development. Resistant viruses first acquired a mutation in, or adjacent to, the α2 helix of 2C. This mutation disrupted compound binding and provided drug resistance, but this was at the cost of viral fitness. Additional mutations at distantly localized 2C residues were then acquired to increase resistance and/or to compensate for the loss of fitness. Using computational methods to identify solvent accessible tunnels near the α2 helix in the EV-A71 and PV 2C crystal structures, a conserved binding pocket of the inhibitors is proposed.

Ensemble-Based Analysis of the Dynamic Allostery in the PSD-95 PDZ3 Domain in Relation to the General Variability of PDZ Structures.

PDZ domains are abundant interaction hubs found in a number of different proteins and they exhibit characteristic differences in their structure and ligand specificity. Their internal dynamics have been proposed to contribute to their biological activity via changes in conformational entropy upon ligand binding and allosteric modulation. Here we investigate dynamic structural ensembles of PDZ3 of the postsynaptic protein PSD-95, calculated based on previously published backbone and side-chain S order parameters. We show that there are distinct but interdependent structural rearrangements in PDZ3 upon ligand binding and the presence of the intramolecular allosteric modulator helix 3. We have also compared these rearrangements in PDZ1-2 of PSD-95 and the conformational diversity of an extended set of PDZ domains available in the PDB database. We conclude that although the opening-closing rearrangement, occurring upon ligand binding, is likely a general feature for all PDZ domains, the conformer redistribution upon ligand binding along this mode is domain-dependent. Our findings suggest that the structural and functional diversity of PDZ domains is accompanied by a diversity of internal motional modes and their interdependence.

Acquired Mutations in BAX Confer Resistance to BH3 Mimetics in Acute Myeloid Leukemia

Acquired Mutations in BAX Confer Resistance to BH3 Mimetics in Acute Myeloid Leukemia

BAX-Mutated Clonal Hematopoiesis in Patients on Long-Term Venetoclax for Relapsed/Refractory Chronic Lymphocytic Leukemia

BAX-Mutated Clonal Hematopoiesis in Patients on Long-Term Venetoclax for Relapsed/Refractory Chronic Lymphocytic Leukemia

Structure of S. pombe telomerase protein Pof8 C-terminal domain is an xRRM conserved among LARP7 proteins

ABSTRACT La-related proteins 7 (LARP7) are a class of RNA chaperones that bind the 3′ ends of RNA and are constitutively associated with their specific target RNAs. In metazoa, Larp7 binds to the long non-coding 7SK RNA as a core component of the 7SK RNP, a major regulator of eukaryotic transcription. In the ciliate Tetrahymena the LARP7 protein p65 is a component of telomerase, an essential ribonucleoprotein complex that maintains the telomeric DNA at eukaryotic chromosome ends. p65 is important for the ordered assembly of telomerase RNA (TER) with telomerase reverse transcriptase. Unexpectedly, Schizosaccharomyces pombe Pof8 was recently identified as a LARP7 protein and a core component of fission yeast telomerase essential for biogenesis. LARP7 proteins have a conserved N-terminal La motif and RRM1 (La module) and C-terminal RRM2 with specific RNA substrate recognition attributed to RRM2, first structurally characterized in p65 as an atypical RRM named xRRM. Here we present the X-ray crystal structure and NMR studies of S. pombe Pof8 RRM2. Sequence and structure comparison of Pof8 RRM2 to p65 and human Larp7 xRRMs reveals conserved features for RNA binding with the main variability in the length of the non-canonical helix α3. This study shows that Pof8 has conserved xRRM features, providing insight into TER recognition and the defining characteristics of the xRRM.

Evaluating Phenyl Propanoids Isolated from Citrus medica as Potential Inhibitors for Mitotic kinesin Eg5

Background:Human mitotic kinesins play an essential role in mitotic cell division. Targeting the spindle separation phase of mitosis has gained much attention in cancer chemotherapy. Spindle segregation is carried out mainly by the kinesin, Eg5. Many Eg5 inhibitors are in different phases of clinical trials as cancer drugs. This enzyme has two allosteric binding sites to which the inhibitors can bind. The first site is formed by loop L5, helix α2 and helix α3 and all the current drug candidates bind un-competitively to this site with ATP/ADP. The second site, formed by helix α4 and helix α6, which has gained attention recently, has not been explored well. Some inhibitors that bind to this site are competitive, while others are uncompetitive to ATP/ADP. Phenylpropanoids are pharmacologically active secondary metabolites.Methods:In this study, we have evaluated fourteen phenyl propanoids extracted from Citrus medica for inhibitory activity against human mitotic kinesin Eg5 in vitro steady-state ATPase assay. Ther interactions and stability using molecular docking and molecular dynamics simulations.Results and Discussions:Of the fourteen compounds tested, naringin and quercetin showed good activity with IC50 values in the micromolar range. Molecular docking studies of these complexes showed that both the molecules interact with the key residues of the active site predominantly thorough hydrophobic & aromatic π–π interactions consistent with the known inhibitors. Besides, these molecules also form hydrogen bonding interactions stabilizing the complexes. Molecular dynamics simulations of these complexes confirm the stability of these interactions.Conclusion:These results can be used as a strong basis for further modification of these compounds to design new inhibitors with higher potency using structure-based drug design.

Reversible autoinhibitory regulation of Escherichia coli metallopeptidase BepA for selective β-barrel protein degradation

Escherichia coli periplasmic zinc-metallopeptidase BepA normally functions by promoting maturation of LptD, a β-barrel outer-membrane protein involved in biogenesis of lipopolysaccharides, but degrades it when its membrane assembly is hampered. These processes should be properly regulated to ensure normal biogenesis of LptD. The underlying mechanism of regulation, however, remains to be elucidated. A recently solved BepA structure has revealed unique features: In particular, the active site is buried in the protease domain and conceivably inaccessible for substrate degradation. Additionally, the His-246 residue in the loop region containing helix α9 (α9/H246 loop), which has potential flexibility and covers the active site, coordinates the zinc ion as the fourth ligand to exclude a catalytic water molecule, thereby suggesting that the crystal structure of BepA represents a latent form. To examine the roles of the α9/H246 loop in the regulation of BepA activity, we constructed BepA mutants with a His-246 mutation or a deletion of the α9/H246 loop and analyzed their activities in vivo and in vitro. These mutants exhibited an elevated protease activity and, unlike the wild-type BepA, degraded LptD that is in the normal assembly pathway. In contrast, tethering of the α9/H246 loop repressed the LptD degradation, which suggests that the flexibility of this loop is important to the exhibition of protease activity. Based on these results, we propose that the α9/H246 loop undergoes a reversible structural change that enables His-246-mediated switching (histidine switch) of its protease activity, which is important for regulated degradation of stalled/misassembled LptD.

Computational characterisation of Toxoplasma gondii FabG (3-oxoacyl-[acyl-carrier-protein] reductase): a combined virtual screening and all-atom molecular dynamics simulation study

Toxoplasma gondii is an opportunistic obligate parasite, ubiquitous around the globe with seropositivity rates that range from 10% to 90% and infection by the parasite of pregnant women causes pre-natal death of the foetus in most cases and severe neurodegenerative syndromes in some. No vaccine is currently available, and since drug-resistance is common among T. gondii strains, discovering lead compounds for drug design using diverse tactics is necessary. In this study, the sole constituent isoform of an enzymatic 3-oxoacyl-[acyl-carrier-protein] reduction step in an apicoplast-located fatty acid biosynthesis pathway was chosen as a possible drug target. FASII is prokaryotic therefore, targeting it would pose fewer side-effects to human hosts. After a homology 3D modelling of TgFabG, a high-throughput virtual screening of 9867 compounds, the elimination of ligands was carried out by a flexible ligand molecular docking and 200 ns molecular dynamics simulations, with additional DCCM and PC plot analyses. Molecular Dynamics and related post-MD analyses of the top 3 TgFabG binders selected for optimal free binding energies, showed that L2 maintained strong H-bonds with TgFabG and facilitated structural reorientation expected of FabGs, namely an expansion of the Rossmann Fold and a flexible lid capping. The most flexible TgFabG sites were the α7 helix (the flexible lid region) and the β4-α4 and β5-α6 loops. For TgFabG-L2, the movements of these regions toward the active site enabled greater ligand stability. Thus, L2 ("Skimmine"; PubChem ID: 320361), was ultimately selected as the optimal candidate for the discovery of lead compounds for rational drug design. Communicated by Ramaswamy H. Sarma

Vaccinia Virus Immunomodulator A46: Destructive Interactions with MAL and MyD88 Shown by Negative-Stain Electron Microscopy

Vaccinia virus A46 is an anti-inflammatory and non-anti-apoptotic, two-domain member of the poxviral Bcl-2-like protein family that inhibits the cellular innate immune response at the level of the Toll/interleukin-1 receptor (TIR) domain-containing TLR adaptor proteins MAL, MyD88, TRAM, and TRIF. The mechanism of interaction of A46 with its targets has remained unclear. The TIR domains of MAL and MyD88 have been shown to signal by forming filamentous assemblies. We show a clear concentration-dependent destruction of both of these assemblies by A46 by means of negative-stain electron microscopy from molar ratios of 1:15 for MAL and 1:30 for MyD88. Using targeted mutagenesis and protein-protein crosslinking, we show that A46 interacts with MAL and MyD88 through several facets, including residues on helices α1 and α7 and the C-terminal flexible region. We propose a model in which A46 targets the MAL and MyD88 signalosome intra-strand interfaces and gradually destroys their assemblies in a concentration-dependent manner.

Revealing secrets of the enigmatic omega subunit of bacterial RNA polymerase.

The conserved omega (ω) subunit of RNA polymerase (RNAP) is the only nonessential subunit of bacterial RNAP core. The small ω subunit (7kDa-11.5kDa) contains three conserved α helices, and helices α2 and α3 contain five fully conserved amino acids of ω. Four conserved amino acids stabilize the correct folding of the ω subunit and one is located in the vicinity of the β' subunit of RNAP. Otherwise ω shows high variation between bacterial taxa, and although the main interaction partner of ω is always β', many interactions are taxon-specific. ω-less strains show pleiotropic phenotypes, and based on in vivo and in vitro results, a few roles for the ω subunits have been described. Interactions of the ω subunit with the β' subunit are important for the RNAP core assembly and integrity. In addition, the ω subunit plays a role in promoter selection, as ω-less RNAP cores recruit fewer primary σ factors and more alternative σ factors than intact RNAP cores in many species. Furthermore, the promoter selection of an ω-less RNAP holoenzyme bearing the primary σ factor seems to differ from that of an intact RNAP holoenzyme.

GTP-State-Selective Cyclic Peptide Ligands of K-Ras(G12D) Block Its Interaction with Raf.

We report the identification of three cyclic peptide ligands of K-Ras(G12D) using an integrated in vitro translation–mRNA display selection platform. These cyclic peptides show preferential binding to the GTP-bound state of K-Ras(G12D) over the GDP-bound state and block Ras-Raf interaction. A co-crystal structure of peptide KD2 with K-Ras(G12D)·GppNHp reveals that this peptide binds in the Switch II groove region with concomitant opening of the Switch II loop and a 40° rotation of the α2 helix, and that a threonine residue (Thr10) on KD2 has direct access to the mutant aspartate (Asp12) on K-Ras. Replacing this threonine with non-natural amino acids afforded peptides with improved potency at inhibiting the interaction between Raf1-RBD and K-Ras(G12D) but not wildtype K-Ras. The union of G12D over wildtype selectivity and GTP state/GDP state selectivity is particularly desirable, considering that oncogenic K-Ras(G12D) exists predominantly in the GTP state in cancer cells, and wildtype K-Ras signaling is important for the maintenance of healthy cells.

Molecular basis of the potential interaction of SARS-CoV-2 spike protein to CD147 in COVID-19 associated-lymphopenia

Lymphopenia is considered one of the most characteristic clinical features of the coronavirus disease 2019 (COVID-19). SARS-CoV-2 infects host cells via the interaction of its spike protein with the human angiotensin-converting enzyme 2 (hACE2) receptor. Since T lymphocytes display a very low expression level of hACE2, a novel receptor might be involved in the entry of SARS-CoV-2 into T cells. The transmembrane glycoprotein CD147 is highly expressed by activated T lymphocytes, and was recently proposed as a probable route for SARS-CoV-2 invasion. To understand the molecular basis of the potential interaction of SARS-CoV-2 to CD147, we have investigated the binding of the viral spike protein to this receptor in-silico. The results showed that this binding is dominated by electrostatic interactions involving residues Arg403, Asn481, and the backbone of Gly502. The overall binding arrangement shows the CD147 C-terminal domain interacting with the spike external subdomain in the grove between the short antiparallel β strands, β1’ and β2’, and the small helix α1’. This proposed interaction was further confirmed using MD simulation and binding free energy calculation. These data contribute to a better understanding of the mechanism of infection of SARS-CoV-2 to T lymphocytes and could provide valuable insights for the rational design of adjuvant treatment for COVID-19. Communicated by Ramaswamy H. Sarma

Agonist Binding and G Protein Coupling in Histamine H2 Receptor: A Molecular Dynamics Study.

The histamine H receptor (H2R) plays an important role in the regulation of gastric acid secretion. Therefore, it is a main drug target for the treatment of gastroesophageal reflux or peptic ulcer disease. However, there is as of yet no 3D-structural information available hampering a mechanistic understanding of H2R. Therefore, we created a model of the histamine-H2R-Gs complex based on the structure of the ternary complex of the -adrenoceptor and investigated the conformational stability of this active GPCR conformation. Since the physiologically relevant motions with respect to ligand binding and conformational changes of GPCRs can only partly be assessed on the timescale of conventional MD (cMD) simulations, we also applied metadynamics and Gaussian accelerated molecular dynamics (GaMD) simulations. A multiple walker metadynamics simulation in combination with cMD was applied for the determination of the histamine binding mode. The preferential binding pose detected is in good agreement with previous data from site directed mutagenesis and provides a basis for rational ligand design. Inspection of the H2R-Gs interface reveals a network of polar interactions that may contribute to H2R coupling selectivity. The cMD and GaMD simulations demonstrate that the active conformation is retained on a s-timescale in the ternary histamine-H2R-Gs complex and in a truncated complex that contains only Gs helix 5 instead of the entire G protein. In contrast, histamine alone is unable to stabilize the active conformation, which is in line with previous studies of other GPCRs.

Lysozyme crystals dyed with bromophenol blue: where has the dye gone?

Protein crystals can easily be coloured by adding dyes to their mother liquor, but most structures of these protein-dye complexes remain unsolved. Here, structures of lysozyme in complex with bromophenol blue obtained by soaking orthorhombic and tetragonal crystals in a saturated solution of the dye at different pH values from 5.0 to 7.5 are reported. Two different binding sites can be found in the lysozyme-bromophenol blue crystals: binding site I is located near the amino- and carboxyl-termini, while binding site II is located adjacent to helices α1 (residues 4-15) and α3 (residues 88-100). In the orthorhombic crystals soaked at pH 7.0, binding of the dye takes place in both sites without significant changes in the unit cell. However, soaking tetragonal crystals with bromophenol blue results in two different complexes. Crystals soaked at pH 5.5 (HEWL-T1) show a single dye molecule bound to site II, and the crystals belong to space group P43212 without significant changes in the unit cell (a = b = 78.50, c= 37.34 Å). On the other hand, crystals soaked at pH 6.5 in the presence of imidazole (HEWL-T2) show up to eight molecules of the dye bound to site II, and display changes in space group (P212121) and unit cell (a = 38.00, b = 76.65, c= 84.86 Å). In all of the structures, the dye molecules are placed at the surface of the protein near to positively charged residues accessible through the main solvent channels of the crystal. Differences in the arrangement of the dye molecules at the surface of the protein suggest that the binding is not specific and is mainly driven by electrostatic interactions.