Structure Of Pocket Research Articles

Source pocket-engineered hetero-gate dielectric SOI Tunnel FET with improved performance

In this paper, we have studied and proposed a planar Tunnel FET device with source side SiGe pocket and hetero-gate dielectric structure that works by utilizing quantum mechanical Band-to-Band tunnelling (BTBT) mechanism. Simulation results obtained using a calibrated SILVACO TCAD device simulator show that the device exhibits good ambipolar performance because of the hetero-gate dielectric and the use of source pocket leads to steep point subthreshold swing of 4.90 mV/dec., an average subthreshold swing of 25.31 mV/dec. and a threshold voltage of 0.23 V. For including the hetero-gate dielectric, the variation in drain current values with high-κ dielectric length is analysed. The change in transfer characteristics with pocket parameters including pocket length and pocket doping is also reported. A 4 nm Si0.7Ge0.3 n+ pocket with doping concentration of 5 × 1019 cm−3 is found to show the maximum ON-OFF current ratio of 5.23 × 1011. This paper also performs a comparative study between different TFET structures and the analysis clearly highlights the potential of proposed Source Pocket-Engineered Hetero Gate Dielectric (SPE-HGD) TFET device for low-power and energy-efficient applications.

Crystal structure and substrate recognition mechanism of the prolyl endoprotease PEP from Aspergillus niger

Proteases are enzymes that are not only essential for life but also industrially important. Understanding the substrate recognition mechanisms of proteases is important to enhance the use of proteases. The fungus Aspergillus produces a wide variety of proteases, including PEP, which is a prolyl endoprotease from A. niger. Although PEP exhibits amino acid sequence similarity to the serine peptidase family S28 proteins (PRCP and DPP7) that recognize Pro-X bonds in the terminal regions of peptides, PEP recognizes Pro-X bonds not only in peptides but also in proteins. To reveal the structural basis of the prolyl endoprotease activity of PEP, we determined the structure of PEP by X-ray crystallography at a resolution of 1.75 Å. The PEP structure shows that PEP has a wide-open catalytic pocket compared to its homologs. The characteristic catalytic pocket structure of PEP is predicted to be important for the recognition of protein substrates.

A Novel Chondroitin AC Lyase With Broad Substrate Specificity From Pedobacter rhizosphaerae: Cloning, Expression, and Characterization.

Chondroitin AC lyase (ChSaseAC) is one of the essential polysaccharides lyases in low molecular chondroitin sulfate production. In this work, a novel PrChSaseAC from Pedobacter rhizosphaerae was successfully cloned, expressed in Escherichia coli. After optimizing the induction, the recombinant PrChSaseAC could be expressed efficiently at 0.1 mM IPTG, 25°C, and 12 h induction. Then, it was purified with Ni-NTA affinity chromatography. The characterization of the purified PrChSaseAC showed that it had high specific activity and good storage stability, which would favor the production of low molecular weight chondroitin sulfate. It also displayed activity toward chondroitin sulfate C and hyaluronic acid. PrChSaseAC had the highest activity at pH 7.5, 37°C, 10 mM Ca2+, and 5 mg/ml of chondroitin sulfate A. Molecular docking of substrate and enzyme showed the interactions between the enzyme and substrate; it revealed that the enzyme showed high activity to CS-A and hyaluronic acid, but lower activity to CS-C attributed to the structure of the binding pocket. The high stability and specific activity of the enzyme will benefit the industrial production or clinical treatment.

The Crystal Structure of Nα-p-tosyl-lysyl Chloromethylketone-Bound Oligopeptidase B from Serratia Proteamaculans Revealed a New Type of Inhibitor Binding

A covalent serine protease inhibitor—Na-p-Tosyl-Lysyl Chloromethylketone (TCK) is a modified lysine residue tosylated at the N-terminus and chloromethylated at the C-terminus, one molecule of which is capable of forming two covalent bonds with both Ser and His catalytic residues, was co-crystallized with modified oligopeptidase B (OpB) from Serratia proteomaculans (PSPmod). The kinetics study, which preceded crystallization, shows that the stoichiometry of TCK-dependent inhibition of PSPmod was 1:2 (protein:inhibitor). The crystal structure of the PSPmod-TCK complex, solved at a resolution of 2.3 Å, confirmed a new type of inhibitor binding. Two TCK molecules were bound to one enzyme molecule: one with the catalytic Ser, the other with the catalytic His. Due to this mode of binding, the intermediate state of PSPmod and the disturbed conformation of the catalytic triad were preserved in the PSPmod-TCK complex. Nevertheless, the analysis of the amino acid surroundings of the inhibitor molecule bound to the catalytic Ser and its comparison with that of antipain-bound OpB from Trypanosoma brucei provided an insight in the structure of the PSPmod substrate-binding pocket. Supposedly, the new type of binding is typical for the interaction of chloromethylketone derivatives with two-domain OpBs. In the open conformational state that these enzymes are assumed in solution, the disordered configuration of the catalytic triad prevents simultaneous interaction of one inhibitor molecule with two catalytic residues.

Synthesis of silicate and alumina pillared MWW zeolite as ethane dehydroaromatization catalyst

Pillared MWW zeolites were prepared with polymeric hydroxyl-aluminum Keggin-Al13 cations and tetraethylorthosilicate as pillaring agents to investigate catalyst acidity and interlayer structure impact on ethane dehydroaromatization. The intercalation effect of the pillars causes the MWW zeolite to be stretched between the layers, which cause the 12 member ring (MR) pocket structure to be exposed to outside surface. The modification method can effectively generate the micropore and mesopore MWW zeolite composite, MCM-36. The introduction of silica columns and alumina columns led to the overall decrease of acidity and the increase of weak and Lewis acid of pillared MWW zeolites, respectively. Although the strong Brönsted acid sites of MWW play major effect on catalyzing aromatization of ethylene generated by the dehydrogenation of ethane to aromatics, the modification decreases the cracking of ethylene oligomers. The 12-MR pockets exposed on the pillared MWW zeolite can provide more reaction sites for xylene generation.

Proposal of selective inhibitor for bacterial zinc metalloprotease: Molecular mechanics and ab initio molecular orbital calculations

The zinc metalloprotease pseudolysin (PLN) secreted from Pseudomonas aeruginosa degrades extracellular proteins to produce bacterial nutrition, and various types of PLN inhibitors have been developed to suppress the bacterial growth. However, as the structure of the ligand-binding pocket of PLN has large similarities to those of human matrix metalloproteinases (MMPs) and other human zinc metalloprotease, there is a risk that PLN inhibitors also inhibit human zinc proteases. In this study, we propose a novel agent that may bind stronger to PLN than to MMPs. The compound is proposed based on the specific molecular interactions between existing agents and PLN/MMP metalloproteases evaluated by the present molecular simulations. First, we confirmed that the binding energies of PLN agents evaluated using the ab initio fragment molecular orbital method were comparable to the IC50 values obtained through previous experiments. In addition, the specific molecular interactions between these agents and MMP-9 were investigated to elucidate the fact that some of the agents bind weaker to MMP than PLN. Based on the results, we proposed a novel agent having a succinimide group introduce by a hydroxamic acid group and investigated its binding properties with PLN and MMP. The results may provide useful information for the development of potent inhibitors for PLN with few potential side effects in human bodies.

Effect of immature tick-borne encephalitis virus particles on antiviral activity of 5-aminoisoxazole-3-carboxylic acid adamantylmethyl esters.

Tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus, is common in Europe and Asia and causes a severe disease of the central nervous system. A promising approach in the development of therapy for TBEV infection is the search for small molecule antivirals targeting the flavivirus envelope protein E, particularly its β-n-octyl-d-glucoside binding pocket (β-OG pocket). However, experimental studies of candidate antivirals may be complicated by varying amounts and different forms of the protein E in the virus samples. Viral particles with different conformations and arrangements of the protein E are produced during the replication cycle of flaviviruses, including mature, partially mature, and immature forms, as well as subviral particles lacking genomic RNA. The immature forms are known to be abundant in the viral population. We obtained immature virion preparations of TBEV, characterized them by RT-qPCR, and assessed in vivo and in vitro infectivity of the residual mature virions in the immature virus samples. Analysis of the β-OG pocket structure on the immature virions confirmed the possibility of binding of adamantylmethyl esters of 5-aminoisoxazole-3-carboxylic acid in the pocket. We demonstrated that the antiviral activity of these compounds in plaque reduction assay is significantly reduced in the presence of immature TBEV particles.

Theoretical study of enantioenriched aminohydroxylation of styrene catalyzed by an engineered hemoprotein

AbstractTransforming olefins to chiral amino alcohols is a useful approach to synthesize biologically active natural products and numerous drugs. A recent study has demonstrated a promising and synthetic value of an engineered hemoprotein for catalyzing olefins to chiral amino alcohols with 2500 total turnover numbers and 90% ee. Density functional theory (DFT) calculation has been used to systematically investigate the detailed mechanisms of the aforementioned process. One electron transfers from Fe atom to HN–nitrene in the iron–nitrene intermediate formation. Subsequently, styrene aziridination, singlet state is characterized by a nonradical, concerted nonsynchronous mechanism, while a radical and stepwise mechanism for triplet. Through hydrolysis reaction forming amino alcohol enantiomers, radical intermediate in triplet state without ring‐opening process is obviously more feasible than singlet aziridine, where the energy barrier difference between triplet and singlet approaches to 20.00 kcal/mol. Moreover, due to the hydrogen bond effect, the water dimer‐assisted hydrolysis reaction is effective to reduce the energy barrier by about 7.00 kcal/mol compared with one water assisted in triplet; however, the energy barrier difference in singlet is unapparent with only 0.18 kcal/mol accompanied with ring‐opening process. Further, homology modeling shows that the reactivity and enantioselectivity can be attributed to the structure of the enzyme active pocket. This study sheds light on the mechanism of engineered hemoprotein‐mediated amino alcohols synthesis and shows the development of biological catalysts.

End-to-end learning for compound activity prediction based on binding pocket information

BackgroundRecently, machine learning-based ligand activity prediction methods have been greatly improved. However, if known active compounds of a target protein are unavailable, the machine learning-based method cannot be applied. In such cases, docking simulation is generally applied because it only requires a tertiary structure of the target protein. However, the conformation search and the evaluation of binding energy of docking simulation are computationally heavy and thus docking simulation needs huge computational resources. Thus, if we can apply a machine learning-based activity prediction method for a novel target protein, such methods would be highly useful. Recently, Tsubaki et al. proposed an end-to-end learning method to predict the activity of compounds for novel target proteins. However, the prediction accuracy of the method was still insufficient because it only used amino acid sequence information of a protein as the input.ResultsIn this research, we proposed an end-to-end learning-based compound activity prediction using structure information of a binding pocket of a target protein. The proposed method learns the important features by end-to-end learning using a graph neural network both for a compound structure and a protein binding pocket structure. As a result of the evaluation experiments, the proposed method has shown higher accuracy than an existing method using amino acid sequence information.ConclusionsThe proposed method achieved equivalent accuracy to docking simulation using AutoDock Vina with much shorter computing time. This indicated that a machine learning-based approach would be promising even for novel target proteins in activity prediction.

The Mpro Structure-Based Modifications of Ebselen Derivatives for Improved Antiviral Activity Against SARS-CoV-2 Virus

The main protease (Mpro or 3CLpro) of SARS-CoV-2 virus is a cysteine enzyme critical for viral replication and transcription, thus indicating a potential target for antiviral therapy. A recent repurposing effort has identified ebselen, a multifunctional drug candidate, as an inhibitor of Mpro. Our docking of ebselen to crystal structure of Mpro catalytic pocket reveals a better fit of “tail-to-head” mode through noncovalent binding, suggesting modification of ebselen for its improvement of potency, antiviral activity and selectivity. To test this hypothesis, we designed and synthesized ebselen derivatives aimed at enhancing their non-covalent bonds within Mpro and reducing steric hindrance. The inhibition of Mpro by ebselen derivatives (0.3 μM) was screened in both HPLC and FRET assays. Nine ebselen derivatives exhibited stronger inhibitory effect on Mpro with IC50 of 0.07 to 0.38 μM. Further evaluation of three derivatives showed the inhibition on SARS-CoV-2 viral replication with their IC50 values from 4.08 to 19.93 µM in HPAepiC cells, as compared to prototype ebselen at 24.61 μM. Taken together, our identification of ebselen derivatives with improved antiviral activity may lead to developmental potential for treatment of COVID-19 and SARS-CoV-2 infection.

Reducing the Drain Leakage Current in a Double-Gate Junctionless MOSFET Using the Electron Screening Effect

This study investigated the position of electrons and holes in the ON and OFF states of double-gate junctionless transistors, and then three structures were proposed to create an electron-filled region exposing the drain-side inside the channel to the electron screening effect. Formation of an electron cloud in the channel and on the drain-side damps the electric field resulting from the drain voltage as the electric field passes through the electron cloud before reaching the main channel. Accordingly, in the structure with a drain-side gate, with the electric field decay introduced by the drain to the main channel, the carrier density increased compared to other structures, eventually reducing drain leakage current and improving the channel length modulation (CLM), threshold voltage, drain induced barrier lowering effect, and subthreshold slope in this structure. In the N+ pocket structure, despite the decrease in the electric field resulting from the drain voltage, the drain leakage current and the CLM were increased. In the N+ SiGe pocket structure, although the electric field resulting from the drain voltage along the channel length was reduced compared to the main structure, the drain leakage current and CLM experienced an increase. Moreover, the results showed that at low drain voltages, the drain leakage current in the N+ pocket structure was higher than that of the N+ SiGe pocket, but at high drain voltages, the order was reversed.

Cu2+ Inhibits the Peroxidase and Antibacterial Activity of Homodimer Hemoglobin From Blood Clam Tegillarca granosa by Destroying Its Heme Pocket Structure

Beyond its role as an oxygen transport protein, the homodimer hemoglobin of blood clam Tegillarca granosa (Tg-HbI) has been found to possess antibacterial activity. However, the mechanism of antibacterial activity of Tg-HbI remain to be investigated. In this study, we investigated the effects of Cu2+ on the structure, peroxidase activity, and antibacterial ability of Tg-HbI. Tg-HbI was significantly inactivated by Cu2+ in a non-competitive inhibition manner, following first-order reaction kinetics. The Spectroscopy results showed that Cu2+ changed the iron porphyrin ring and the coordination of heme with proximal histidine of Tg-HbI, and increased the hydrophobicity of heme pocket. We found that proline could stabilize the heme pocket structure of Tg-HbI, hence, protect peroxidase activity and antimicrobial activity of Tg-HbI against damage by Cu2+. Our results suggest that Cu2+ inhibits the peroxidase and antibacterial activity of Tg-HbI by destroying its heme pocket structure and Tg-HbI probably plays an antibacterial role through its peroxidase activity. This result could provide insights into the antibacterial mechanism of Tg-HbI.

Analyzing Kinase Similarity in Small Molecule and Protein Structural Space to Explore the Limits of Multi-Target Screening.

While selective inhibition is one of the key assets for a small molecule drug, many diseases can only be tackled by simultaneous inhibition of several proteins. An example where achieving selectivity is especially challenging are ligands targeting human kinases. This difficulty arises from the high structural conservation of the kinase ATP binding sites, the area targeted by most inhibitors. We investigated the possibility to identify novel small molecule ligands with pre-defined binding profiles for a series of kinase targets and anti-targets by in silico docking. The candidate ligands originating from these calculations were assayed to determine their experimental binding profiles. Compared to previous studies, the acquired hit rates were low in this specific setup, which aimed at not only selecting multi-target kinase ligands, but also designing out binding to anti-targets. Specifically, only a single profiled substance could be verified as a sub-micromolar, dual-specific EGFR/ErbB2 ligand that indeed avoided its selected anti-target BRAF. We subsequently re-analyzed our target choice and in silico strategy based on these findings, with a particular emphasis on the hit rates that can be expected from a given target combination. To that end, we supplemented the structure-based docking calculations with bioinformatic considerations of binding pocket sequence and structure similarity as well as ligand-centric comparisons of kinases. Taken together, our results provide a multi-faceted picture of how pocket space can determine the success of docking in multi-target drug discovery efforts.

A Perspective on Natural and Nature-Inspired Small Molecules Targeting Phosphodiesterase 9 (PDE9): Chances and Challenges against Neurodegeneration.

As life expectancy increases, dementia affects a growing number of people worldwide. Besides current treatments, phosphodiesterase 9 (PDE9) represents an alternative target for developing innovative small molecules to contrast neurodegeneration. PDE inhibition promotes neurotransmitter release, amelioration of microvascular dysfunction, and neuronal plasticity. This review will provide an update on natural and nature-inspired PDE9 inhibitors, with a focus on the structural features of PDE9 that encourage the development of isoform-selective ligands. The expression in the brain, the presence within its structure of a peculiar accessory pocket, the asymmetry between the two subunits composing the protein dimer, and the selectivity towards chiral species make PDE9 a suitable target to develop specific inhibitors. Additionally, the world of natural compounds is an ideal source for identifying novel, possibly asymmetric, scaffolds, and xanthines, flavonoids, neolignans, and their derivatives are currently being studied. In this review, the available literature data were interpreted and clarified, from a structural point of view, taking advantage of molecular modeling: 3D structures of ligand-target complexes were retrieved, or built, and discussed.

MYC inhibitors in multiple myeloma.

The importance of MYC function in cancer was discovered in the late 1970s when the sequence of the avian retrovirus that causes myelocytic leukemia was identified. Since then, over 40 years of unceasing research have highlighted the significance of this protein in malignant transformation, especially in hematologic diseases. Indeed, some of the earliest connections among the higher expression of proto-oncogenes (such as MYC), genetic rearrangements and their relation to cancer development were made in Burkitt lymphoma, chronic myeloid leukemia and mouse plasmacytomas. Multiple myeloma (MM), in particular, is a plasma cell malignancy strictly associated with MYC deregulation, suggesting that therapeutic strategies against it would be beneficial in treating this disease. However, targeting MYC was - and, somehow, still is - challenging due to its unique properties: lack of defined three-dimensional structure, nuclear localization and absence of a targetable enzymatic pocket. Despite these difficulties, however, many studies have shown the potential therapeutic impact of direct or indirect MYC inhibition. Different molecules have been tested, in fact, in the context of MM. In this review, we summarize the current status of the different compounds, including the results of their clinical testing, and propose to continue with the efforts to identify, repurpose, redesign or improve drug candidates to combine them with standard of care therapies to overcome resistance and enable better management of myeloma treatment.

Endoscopic endonasal reconstruction of skull base defects in the lateral recess of the sphenoid sinus: evaluation of computed tomograms for planning operations

Determine the influence of the anatomical features and sizes of the lateral pocket with a defect on the choice of surgical access and the quality of the performed plastics. A retrospective analysis of computed tomograms of 38 patients who underwent surgical treatment at the Burdenko National Medical Research Center for Neurosurgery of the Ministry of Health of Russia about defects of the skull base in the area of the lateral pocket of the sphenoid sinus. The patients were divided into three groups depending on the approach used (the 1st group), the recurrence rate (the 2nd group), and the characteristics of intraoperative visualization of the defect (the 3rd group). There were no statistically significant differences in anatomical features in patients who underwent trans-pterygoid and transsphenoidal approaches, as well as in patients of the 2nd group. Patients of the 3rd group (with visualization features) showed statistically significant differences between the distance from the defect to the VR line (p=0.007). In patients with good visualization of the defect using direct optics, this distance was shorter than in patients in whom the defect was visualized with an angled endoscope. No other anatomical differences were noted. The anatomical features of the lateral pocket structure (type of pneumatization, size and volume) did not affect the choice of access to the defect and the frequency of recurrence. When comparing the approaches, it was noted that the trans-pterygoid access, providing direct visualization of defects, minimizes the risk of recurrence in the postoperative period. An objective anatomical indicator for choosing an access to the defects of the lateral pocket can be the distance from the defect to the VR line: at a distance of more than 0.7 cm, it is advisable to perform a trans-pterygoid approach; at a distance of less than 0.7 cm, it is possible to achieve direct visualization of the defect and perform high-quality plastic surgery with a transsphenoidal access.

Insights into the dynamics of cyclic diguanosine monophosphate I riboswitch using molecular dynamics simulation

Riboswitches are key cis-regulatory elements, present at 5' UTRs of several prokaryotic mRNAs, involved in gene expression regulation by binding selectively to specific ligands followed by conformational changes. However, understanding of ligand-free riboswitch, conformational changes between the ligand-free and ligand-bound riboswitch and binding mechanism of ligand to the aptamer of riboswitch is limited. In the present paper we describe the structural and dynamical properties of ligand-free c-di-GMP I riboswitch aptamer and its possible binding mechanism with c-di-GMP by means of all-atom molecular dynamics (MD) simulations. Various analyses such as principal component analysis, cross correlation dynamics analysis, network analysis and trajectory analyses were carried out. The ligand-free structure shows stable conformation with folded P2, P3 and an unwind P1 helix with open binding pocket at helix-join-helix junction while the ligand-bound structure showed closed binding pocket structure compared to the ligand-free structure. The junction residues significantly showed anti-correlations with P1 helix and weak correlated motions with each other in the open conformation of the ligand-free aptamer of riboswitch. The networks analysis of binding pocket residues suggested interaction among the identified key residues of the binding pocket (G20, A47, C92) and P1 helix illustrating the role of P1 helix in binding of ligand. The structural insights, on c-di-GMP I riboswitch, presented in this paper can be useful for the development of therapeutics against V. cholerae. The understanding of the c-di-GMP I riboswitch at dynamic molecular level provides a potential solution for riboswitch drug design.

Bioinformatics prediction of B and T cell epitopes within the spike and nucleocapsid proteins of SARS-CoV2

BackgroundThe striking difference in severity of SARS CoV2 infection among global population is partly attributed to viral factors. With the spike (S) and nucleocapsid (N) are the most immunogenic subunits, genetic diversity and antigenicity of S and N are key players in virulence and in vaccine development. AimThis paper aims at identifying immunogenic targets for better vaccine development and/or immunotherapy of COVID 19 pandemic. Methods18 complete genomes of SARS CoV2 (n=14), SARS CoV (n=2) and MERS CoV (n=2) were examined. Bioinformatics of viral genetics and protein folding allowed functional tuning of NH2 Terminal Domain (NTD) of S protein and development of epitope maps for B and T cell responses. ConclusionA deletion of amino acid residues Y144 and G107 were discovered in NTD of S protein derived from Indian and French isolates resulting in altered pocket structure exclusively located in NTD and reduced affinity of NTD binding to endogenous nAbs and disrupted NTD mediated cell entry. We therefore, proposed a set of B and T cell epitopes based on Immune Epitope Database, homologous epitopes for nAbs in convalescent plasma post SARS CoV infection and functional domains of S (NTD, Receptor Binding domain and the unique polybasic Furin cleavage site at S1/S2 junction). Nevertheless, laboratory data are required to develop vaccine and immunotherapeutics.

Investigation on the boundary layer transition with the effects of periodic passing wakes

The boundary layer transition caused by wake is a problem related to basic fluid mechanics and engineering applications. In this paper, the interaction between the periodic passing wakes induced by moving cylinders and the boundary layer of the plate is investigated by large eddy simulation, and experiments in a low-speed water tunnel are designed to verify the simulations. The flow field velocity is measured by high-resolution pressure sensors and two-dimensional particle image velocimetry. The effects of wake passing frequencies fN = 0.63, 1, and 1.26 on the time average and statistical average characteristics of the boundary layer transition and the instantaneous flow structure are studied. The influence of large-scale wakes on the integral parameters of boundary layer thickness, skin friction coefficients, time-averaged velocity distributions, and velocity fluctuations is addressed. The results show that the onset of transition moves to the leading edge of the plate as the wake passing frequency increases, while the location of transition completion moves to the end of the plate. The specific boundary layer transition process is analyzed through the spanwise pocket and streamwise streaky structure propagation. The vortex structures in the boundary layer are extracted by the Q criterion, and the results show that the spanwise secondary vortex on the pressure side induced by the large-scale wake gradually loses its stability and results in transition. Moreover, the hairpin vortex in the suction surface continually lifts up the wall-normal location of the breakdown event. Thus, it turns the turbulence spot arrowhead pointing downstream into pointing upstream.

A Not Obvious Correlation Between the Structure of Green Fluorescent Protein Chromophore Pocket and Hydrogen Bond Dynamics: A Choreography From ab initio Molecular Dynamics.

The Green Fluorescent Protein (GFP) is a widely studied chemical system both for its large amount of applications and the complexity of the excited state proton transfer responsible of the change in the protonation state of the chromophore. A detailed investigation on the structure of the chromophore environment and the influence of chromophore form (either neutral or anionic) on it is of crucial importance to understand how these factors could potentially influence the protein function. In this study, we perform a detailed computational investigation based on the analysis of ab-initio molecular dynamics simulations, to disentangle the main structural quantities determining the fine balance in the chromophore environment. We found that specific hydrogen bonds interactions directly involving the chromophore (or not), are correlated to quantities, such as the volume of the cavity in which the chromophore is embedded and that it is importantly affected by the chromophore protonation state. The cross-correlation analysis performed on some of these hydrogen bonds and the cavity volume, demonstrates a direct correlation among them and we also identified the ones specifically involved in this correlation. We also found that specific interactions among residues far in the space are correlated, demonstrating the complexity of the chromophore environment and that many structural quantities have to be taken into account to properly describe and understand the main factors tuning the active site of the protein. From an overall evaluation of the results obtained in this work, it is shown that the residues which a priori are perceived to be spectators play instead an important role in both influencing the chromophore environment (cavity volume) and its dynamics (cross-correlations among spatially distant residues).