Computational Alanine Scanning Mutagenesis Research Articles

Evaluation of interactions between the hepatitis C virus NS3/4A and sulfonamidobenzamide based molecules using molecular docking, molecular dynamics simulations and binding free energy calculations.

The Hepatitis C Virus (HCV) NS3/4A is an attractive target for the treatment of Hepatitis C infection. Herein, we present an investigation of HCV NS3/4A inhibitors based on a sulfonamidobenzamide scaffold. Inhibitor interactions with HCV NS3/4A were explored by molecular docking, molecular dynamics simulations, and MM/PBSA binding free energy calculations. All of the inhibitors adopt similar molecular docking poses in the catalytic site of the protease that are stabilized by hydrogen bond interactions with G137 and the catalytic S139, which are known to be important for potency and binding stability. The quantitative assessments of binding free energies from MM/PBSA correlate well with the experimental results, with a high coefficient of determination, R2 of 0.92. Binding free energy decomposition analyses elucidate the different contributions of Q41, F43, H57, R109, K136, G137, S138, S139, A156, M485, and Q526 in binding different inhibitors. The importance of these sidechain contributions was further confirmed by computational alanine scanning mutagenesis. In addition, the sidechains of K136 and S139 show crucial but distinct contributions to inhibitor binding with HCV NS3/4A. The structural basis of the potency has been elucidated, demonstrating the importance of the R155 sidechain conformation. This extensive exploration of binding energies and interactions between these compounds and HCV NS3/4A at the atomic level should benefit future antiviral drug design.

Enabling biocatalysis in high-concentration organic cosolvent by enzyme gate engineering.

Biocatalysis in high-concentration organic solvents (OSs) offers many advantages, but realizing this process remains a huge challenge. An R-selective ω-amine transaminase variant (AcATAM2 ) exhibited high activity toward 50 g/L pro-sitagliptin ketone 1-[1-piperidinyl]-4-[2,4,5-trifluorophenyl]-1,3-butanedione (PTfpB). However, AcATAM2 displayed unsatisfactory organic-cosolvent resistance against high-concentration dimethyl sulfoxide (DMSO), which is required to enhance the solubility of the hydrophobic substrate PTfpB. Located in the substrate-binding tunnel, enzyme gates are structural elements that undergo reversible conformational transitions, thus affecting the accessibility of the binding pocket to solvent molecules. Depending on the conformation of the enzyme gates, one can define an open or closed conformation on which the enzyme activity in OSs may depend. To enhance the DMSO resistance of AcATAM2 , we identified the beneficial residues at the "enzyme gate" region via computational analysis, alanine scanning, and site-saturation mutagenesis. Two beneficial variants, namely, AcATAM2F56D and AcATAM2F56V , not only displayed improved enzyme activity but also exhibited enhanced DMSO resistance (the half-life value increased from 25.71 to 42.49 h under 60% DMSO). Molecular dynamic simulations revealed that the increase in DMSO resistance was mainly caused by the decrease in the number of DMSO molecules in the substrate-binding pocket. Moreover, in the kilogram-scale experiment, the conversion of 80 g/L substrate was increased from 50% (AcATAM2 ) to 85% (M2F56D in 40% DMSO) with a high e.e. of >99% within 24 h.

Computational Hot-Spot Analysis of the SARS-CoV-2 Receptor Binding Domain/ACE2 Complex*.

Infection and replication of SARS CoV-2 (the virus that causes COVID-19) requires entry to the interior of host cells. In humans, a protein-protein interaction (PPI) between the SARS CoV-2 receptor-binding domain (RBD) and the extracellular peptidase domain of ACE2 on the surface of cells in the lower respiratory tract is an initial step in the entry pathway. Inhibition of the SARS CoV-2 RBD/ACE2 PPI is currently being evaluated as a target for therapeutic and/or prophylactic intervention. However, relatively little is known about the molecular underpinnings of this complex. Employing multiple computational platforms, we predicted "hot-spot" residues in a positive-control PPI (PMI/MDM2) and the CoV-2 RBD/ACE2 complex. Computational alanine scanning mutagenesis was performed to predict changes in Gibbs' free energy that are associated with mutating residues at the positive control (PMI/MDM2) or SARS RBD/ACE2 binding interface to alanine. Additionally, we used the Adaptive Poisson-Boltzmann Solver to calculate macromolecular electrostatic surfaces at the interface of the positive-control PPI and SARS CoV-2/ACE2 PPI. Finally, a comparative analysis of hot-spot residues for SARS-CoV and SARS-CoV-2, in complex with ACE2, is provided. Collectively, this study illuminates predicted hot-spot residues, and clusters, at the SARS CoV-2 RBD/ACE2 binding interface, potentially guiding the development of reagents capable of disrupting this complex and halting COVID-19.

Computational Investigation of Structural Dynamics of SARS-CoV-2 Methyltransferase-Stimulatory Factor Heterodimer nsp16/nsp10 Bound to the Cofactor SAM.

Recently, a highly contagious novel coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, has emerged, posing a global threat to public health. Identifying a potential target and developing vaccines or antiviral drugs is an urgent demand in the absence of approved therapeutic agents. The 5′-capping mechanism of eukaryotic mRNA and some viruses such as coronaviruses (CoVs) are essential for maintaining the RNA stability and protein translation in the virus. SARS-CoV-2 encodes S-adenosyl-L-methionine (SAM) dependent methyltransferase (MTase) enzyme characterized by nsp16 (2′-O-MTase) for generating the capped structure. The present study highlights the binding mechanism of nsp16 and nsp10 to identify the role of nsp10 in MTase activity. Furthermore, we investigated the conformational dynamics and energetics behind the binding of SAM to nsp16 and nsp16/nsp10 heterodimer by employing molecular dynamics simulations in conjunction with the Molecular Mechanics Poisson-Boltzmann Surface Area (MM/PBSA) method. We observed from our simulations that the presence of nsp10 increases the favorable van der Waals and electrostatic interactions between SAM and nsp16. Thus, nsp10 acts as a stimulator for the strong binding of SAM to nsp16. The hydrophobic interactions were predominately identified for the nsp16-nsp10 interactions. Also, the stable hydrogen bonds between Ala83 (nsp16) and Tyr96 (nsp10), and between Gln87 (nsp16) and Leu45 (nsp10) play a vital role in the dimerization of nsp16 and nsp10. Besides, Computational Alanine Scanning (CAS) mutagenesis was performed, which revealed hotspot mutants, namely I40A, V104A, and R86A for the dimer association. Hence, the dimer interface of nsp16/nsp10 could also be a potential target in retarding the 2′-O-MTase activity in SARS-CoV-2. Overall, our study provides a comprehensive understanding of the dynamic and thermodynamic process of binding nsp16 and nsp10 that will contribute to the novel design of peptide inhibitors based on nsp16.

Structural models of human ACE2 variants with SARS-CoV-2 Spike protein for structure-based drug design

Emergence of coronaviruses poses a threat to global health and economy. The current outbreak of SARS-CoV-2 has infected more than 28,000,000 people and killed more than 915,000. To date, there is no treatment for coronavirus infections, making the development of therapies to prevent future epidemics of paramount importance. To this end, we collected information regarding naturally-occurring variants of the Angiotensin-converting enzyme 2 (ACE2), an epithelial receptor that both SARS-CoV and SARS-CoV-2 use to enter the host cells. We built 242 structural models of variants of human ACE2 bound to the receptor binding domain (RBD) of the SARS-CoV-2 surface spike glycoprotein (S protein) and refined their interfaces with HADDOCK. Our dataset includes 140 variants of human ACE2 representing missense mutations found in genome-wide studies, 39 mutants with reported effects on the recognition of the RBD, and 63 predictions after computational alanine scanning mutagenesis of ACE2-RBD interface residues. This dataset will help accelerate the design of therapeutics against SARS-CoV-2, as well as contribute to prevention of possible future coronaviruses outbreaks.

Computational Prediction of Mutational Effects on SARS-CoV-2 Binding by Relative Free Energy Calculations.

The ability of coronaviruses to infect humans is invariably associated with their binding strengths to human receptor proteins. Both SARS-CoV-2, initially named 2019-nCoV, and SARS-CoV were reported to utilize angiotensin-converting enzyme 2 (ACE2) as an entry receptor in human cells. To better understand the interplay between SARS-CoV-2 and ACE2, we performed computational alanine scanning mutagenesis on the “hotspot” residues at protein–protein interfaces using relative free energy calculations. Our data suggest that the mutations in SARS-CoV-2 lead to a greater binding affinity relative to SARS-CoV. In addition, our free energy calculations provide insight into the infectious ability of viruses on a physical basis and also provide useful information for the design of antiviral drugs.

BAlaS: fast, interactive and accessible computational alanine-scanning using BudeAlaScan.

In experimental protein engineering, alanine-scanning mutagenesis involves the replacement of selected residues with alanine to determine the energetic contribution of each side chain to forming an interaction. For example, it is often used to study protein-protein interactions. However, such experiments can be time-consuming and costly, which has led to the development of programmes for performing computational alanine-scanning mutagenesis (CASM) to guide experiments. While programmes are available for this, there is a need for a real-time web application that is accessible to non-expert users. Here, we present BAlaS, an interactive web application for performing CASM via BudeAlaScan and visualizing its results. BAlaS is interactive and intuitive to use. Results are displayed directly in the browser for the structure being interrogated enabling their rapid inspection. BAlaS has broad applications in areas, such as drug discovery and protein-interface design. BAlaS works on all modern browsers and is available through the following website: https://balas.app. The project is open source, distributed using an MIT license and is available on GitHub (https://github.com/wells-wood-research/balas).

Molecular insight into multiple RpoB clinical mutants of Mycobacterium tuberculosis: An attempt to probe structural variations in rifampicin binding site underlying drug resistance

Rifampicin (RMP) resistant strains are still persisting as a threat to the global TB control program. Therefore, understanding the RMP resistant mechanism is need of the hour. The current study has investigated the role of each RMP binding site (RBS) residues, deploying computational alanine scanning mutagenesis (CASM) to unravel the critical and non-critical binding site residues. In addition, conformational shifts in RBS cavity of different RNAP β-subunit (RpoB) systems have also been analyzed. Our initial findings showed that in addition to reported mutational sites, Q510, Q513, R529, P564 and P566 were also critical binding site residues, which upon mutation destabilize the RMP binding. Study also indicated that R3, R4 and R5 regions of RpoB were very significant for the functioning of RMP. The concerted interactions of these regions with RMP hold it into the centre position of RBS and restrict the entrance of elongating RNA transcript. Whereas, after mutation, due to repositioning of RMP and changes in its interactions, the overall cavity becomes significantly hollow which may confer space for phosphodiester bond formation during transcription elongation. Our findings provide valuable details to forestall RMP resistance and may help in the development of new leads against the RMP resistant strains.

Modulation of lateral and longitudinal interdimeric interactions in microtubule models by Laulimalide and Peloruside A association: A molecular modeling approach on the mechanism of microtubule stabilizing agents.

Laulimalide (LAU) and Peloruside A (PLA) are non-taxane microtubule stabilizing agents with promising antimitotic properties. These ligands promote the assembly of microtubules (MTs) by targeting a unique binding site on β-tubulin. The X-ray structure for LAU/PLA-tubulin association was recently elucidated, but little information is available regarding the role of these ligands as modulators of interdimeric interactions across MTs. Herein, we report the use of molecular dynamics (MD), principal component analysis (PCA), MM/GBSA-binding free energy calculations, and computational alanine scanning mutagenesis (ASM) to examine effect of LAU/PLA association on lateral and longitudinal contacts between tubulin dimers in reduced MT models. MD and PCA results revealed that LAU/PLA exerts a strong restriction of lateral and longitudinal interdimeric motions, thus enabling the stabilization of the MT lattice. Besides structural effects, LAU/PLA induces a substantial strengthening of longitudinal interdimeric interactions, whereas lateral contacts are less affected by these ligands, as revealed by MM/GBSA and ASM calculations. These results are valuable to increase understanding about the molecular features involved in MT stabilization by LAU/PLA, and to design novel compounds capable of emulating the mode of action of these ligands.

Properties that rank protein:protein docking poses with high accuracy.

The development of docking algorithms to predict near-native structures of protein:protein complexes from the structure of the isolated monomers is of paramount importance for molecular biology and drug discovery. In this study, we assessed the capacity of the interfacial area of protein:protein complexes and of Molecular Mechanics-Poisson Boltzmann Surface Area (MM-PBSA)-derived properties, to rank docking poses. We used a set of 48 protein:protein complexes, and a total of 67 docking experiments distributed among bound:bound, bound:unbound, and unbound:unbound test cases. The MM-PBSA binding free energy of protein monomers has been shown to be very convenient to predict high-quality structures with a high success rate. In fact, considering solely the top-ranked pose of more than 200 docking solutions of each of 39 protein:protein complexes, the success rate was 77% in the prediction of high-quality poses, or 90% if considering high- or medium-quality poses. If considering high- or medium-quality poses as the top-one prediction, a success rate of 87% was obtained for a scoring scheme based on computational alanine scanning mutagenesis data. Such ranking accuracy highlights the ability of these properties to predict near-native poses in protein:protein docking.

Facile Affinity Maturation of Antibody Variable Domains Using Natural Diversity Mutagenesis.

The identification of mutations that enhance antibody affinity while maintaining high antibody specificity and stability is a time-consuming and laborious process. Here, we report an efficient methodology for systematically and rapidly enhancing the affinity of antibody variable domains while maximizing specificity and stability using novel synthetic antibody libraries. Our approach first uses computational and experimental alanine scanning mutagenesis to identify sites in the complementarity-determining regions (CDRs) that are permissive to mutagenesis while maintaining antigen binding. Next, we mutagenize the most permissive CDR positions using degenerate codons to encode wild-type residues and a small number of the most frequently occurring residues at each CDR position based on natural antibody diversity. This mutagenesis approach results in antibody libraries with variants that have a wide range of numbers of CDR mutations, including antibody domains with single mutations and others with tens of mutations. Finally, we sort the modest size libraries (~10 million variants) displayed on the surface of yeast to identify CDR mutations with the greatest increases in affinity. Importantly, we find that single-domain (VHH) antibodies specific for the α-synuclein protein (whose aggregation is associated with Parkinson’s disease) with the greatest gains in affinity (>5-fold) have several (four to six) CDR mutations. This finding highlights the importance of sampling combinations of CDR mutations during the first step of affinity maturation to maximize the efficiency of the process. Interestingly, we find that some natural diversity mutations simultaneously enhance all three key antibody properties (affinity, specificity, and stability) while other mutations enhance some of these properties (e.g., increased specificity) and display trade-offs in others (e.g., reduced affinity and/or stability). Computational modeling reveals that improvements in affinity are generally not due to direct interactions involving CDR mutations but rather due to indirect effects that enhance existing interactions and/or promote new interactions between the antigen and wild-type CDR residues. We expect that natural diversity mutagenesis will be useful for efficient affinity maturation of a wide range of antibody fragments and full-length antibodies.

Residues of Alpha Helix H3 Determine Distinctive Features of Transforming Growth Factor β3

Transforming growth factors (TGF-βs) are proteins that regulate cell growth by binding to their receptors. In contrast to transforming growth factor (TGF) β1, TGF-β3 homodimer is believed to exist also in an open conformation, in which both of its monomers are loosely packed against each other. At the origin of this difference is the H3-helix. Its sequence and degree of structuration seem to govern the outcome of TGF dimerization. We docked two monomers of TGF-β3 with intact and altered H3 α-helix against each other using HADDOCK. TGF-β3 monomer with an intact H3-helix exclusively forms closed conformations of homodimer, whereas the open conformation may coexist with the closed one when a part of the H3 α-helix is destabilized. We quantify the difference in its conformational preference for the open versus the closed structure by calculating the binding energy between monomers using the MMPBSA approach. We compare the wild type (wt) TGFβ3/TGFβ1 homodimers in the Protein Data Bank to a swapped mutant where all residues of the H3-helix were mutated to the respective TGFβ1/TGFβ3 sequence. Swapping stabilizes the closed conformation and destabilizes the open conformation of TGFβ3. Further detailed insight is derived from molecular dynamics simulation studies suggesting that Val 61 of the H3-helix may act as an anchor residue for the closed conformation of TGFβ3. Computational alanine scanning mutagenesis confirms that several residues of the H3-helix are the hot residues for the closed conformation of TGFβ3. These observations may bear relevance to general conformational transitions in proteins and specifically in the TGFβ superfamily.

Discovering key residues of dengue virus NS2b-NS3-protease: New binding sites for antiviral inhibitors design

The NS2B-NS3 protease is essential for the Dengue Virus (DENV) replication process.This complex constitutes a target for efficient antiviral discovery because a drug could inhibit the viral polyprotein processing. Furthermore, since the protease is highly conserved between the four Dengue virus serotypes, it is probable that a drug would be equally effective against all of them. In this article, a strategy is reported that allowed us to identify influential residues on the function of the Dengue NS2b-NS3 Protease. Moreover, this is a strategy that could be applied to virtually any protein for the search of alternative influential residues, and for non-competitive inhibitor development. First, we incorporated several features derived from computational alanine scanning mutagenesis, sequence, structure conservation, and other structure-based characteristics. Second, these features were used as variables to obtain a multilayer perceptron model to identify defined groups (clusters) of key residues as possible candidate pockets for binding sites of new leads on the DENV protease. The identified residues included: i) amino acids close to the beta sheet-loop-beta sheet known to be important in its closed conformation for NS2b ii) residues close to the active site, iii) several residues evenly spread on the NS2b-NS3 contact surface, and iv) some inner residues most likely related to the overall stability of the protease. In addition, we found concordance on our list of residues with previously identified amino acids part of a highly conserved peptide studied for vaccine development.

Structure and sequence based functional annotation of Zika virus NS2b protein: Computational insights

While Zika virus (ZIKV) outbreaks are a growing concern for global health, a deep understanding about the virus is lacking. Here we report a contribution to the basic science on the virus— a detailed computational analysis of the non structural protein NS2b. This protein acts as a cofactor for the NS3 protease (NS3Pro) domain that is important on the viral life cycle, and is an interesting target for drug development. We found that ZIKV NS2b cofactor is highly similar to other virus within the Flavivirus genus, especially to West Nile Virus, suggesting that it is completely necessary for the protease complex activity. Furthermore, the ZIKV NS2b has an important role to the function and stability of the ZIKV NS3 protease domain even when presents a low conservation score. In addition, ZIKV NS2b is mostly rigid, which could imply a non dynamic nature in substrate recognition. Finally, by performing a computational alanine scanning mutagenesis, we found that residues Gly 52 and Asp 83 in the NS2b could be important in substrate recognition.

Bioinspired Design and Oriented Synthesis of Immunogenic Site-Specifically Penicilloylated Peptides.

β-Lactam antibiotics allergy is recognized as a public health concern. By covalently binding to serum proteins, penicillins are known to form immunogenic complexes. The latter are recognized and digested by antigen-presenting cells into drug-hapten peptides leading to the immunization of treated persons and IgE-mediated hypersensitivity reactions encompassing anaphylaxis. If type I allergic reactions to drugs are often unpredictable, they are known to be dependent on CD4+ T-cells. This fundamental study revisits the chemical basis of the benzylpenicillin (BP) allergy with the aim of identifying immunologically relevant biomimetic benzylpenicilloylated peptides through the analysis of BP-conjugated human serum albumin (BP-HSA) profile and the evaluation of the naı̈ve CD4+ T-cell responses to candidate BP-HSA-derived peptides. The chemical structures of BP-HSA bioconjugates synthesized in vitro at both physiological and basic pH were investigated by mass spectrometry. From the ten most representative lysine residues grafted by BP-hapten, HSA-bioinspired 15-mer peptide sequences were designed and the potential T-cell epitope profile of each peptide was predicted using two complementary in silico approaches, i.e., HLA class II binding prediction tools from the Immune Epitope Database and Analysis Resource (IEDB) and computational alanine scanning mutagenesis. Twelve structurally diversified benzylpenicilloylated peptides (BP-Ps) were selected and synthesized with the aid of a flexible synthesis pathway using an original benzylpenicilloylated lysine monomer as common precursor. In order to corroborate their predicted "epitope" profile, the naı̈ve CD4+ T-cell response specific to BP was evaluated through a coculture approach. To our knowledge, this study showed for the first time the ability of bioinspired peptides structurally stemming from BP-HSA to be recognized by naı̈ve CD4+ T-cells thus identifying a pre-existing T-cell repertoire for penicillin molecules bound to proteins. It also established a promising model approach expandable to other most frequently used penicillin classes of antibiotics to reveal biomimetic drug-modified antigenic peptides relevant for qualitative and quantitative drug allergy studies.

Two common structural motifs for TCR recognition by staphylococcal enterotoxins

Superantigens are toxins produced by Staphylococcus aureus, called staphylococcal enterotoxins (abbreviated SEA to SEU). They can cross-link the T cell receptor (TCR) and major histocompatibility complex class II, triggering a massive T cell activation and hence disease. Due to high stability and toxicity, superantigens are potential agents of bioterrorism. Hence, antagonists may not only be useful in the treatment of disease but also serve as countermeasures to biological warfare. Of particular interest are inhibitors against SEA and SEB. SEA is the main cause of food poisoning, while SEB is a common toxin manufactured as a biological weapon. Here, we present the crystal structures of SEA in complex with TCR and SEE in complex with the same TCR, complemented with computational alanine-scanning mutagenesis of SEA, SEB, SEC3, SEE, and SEH. We have identified two common areas that contribute to the general TCR binding for these superantigens. This paves the way for design of single antagonists directed towards multiple toxins.

Structural insights into Cydia pomonella pheromone binding protein 2 mediated prediction of potentially active semiochemicals.

Given the advantages of behavioral disruption application in pest control and the damage of Cydia pomonella, due progresses have not been made in searching active semiochemicals for codling moth. In this research, 31 candidate semiochemicals were ranked for their binding potential to Cydia pomonella pheromone binding protein 2 (CpomPBP2) by simulated docking, and this sorted result was confirmed by competitive binding assay. This high predicting accuracy of virtual screening led to the construction of a rapid and viable method for semiochemicals searching. By reference to binding mode analyses, hydrogen bond and hydrophobic interaction were suggested to be two key factors in determining ligand affinity, so is the length of molecule chain. So it is concluded that semiochemicals of appropriate chain length with hydroxyl group or carbonyl group at one head tended to be favored by CpomPBP2. Residues involved in binding with each ligand were pointed out as well, which were verified by computational alanine scanning mutagenesis. Progress made in the present study helps establish an efficient method for predicting potentially active compounds and prepares for the application of high-throughput virtual screening in searching semiochemicals by taking insights into binding mode analyses.

Molecular basis of R294K mutation effects of H7N9 neuraminidases with drugs and cyclic peptides: an in silico and experimental study

An overview of Shanghai N9/cyclic peptide I complex structure.

Discovery of new druggable sites in the anti-cholesterol target HMG-CoA reductase by computational alanine scanning mutagenesis

The enzyme 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA-R) is the fundamental target for the treatment of hypercholesterolemia nowadays. The HMG-CoA-R clinical active site inhibitors (statins) are among the most widespread and profitable drugs ever sold but their side effects (myopathies, sometimes severe) still limit their use, which makes the finding of alternatives to statins a field of intense research. In this line, we address here a new strategy for inhibiting the homotetrameric HMG-CoA-R. The enzyme consists of a "dimer of dimers", each dimer having two active sites. We pursue here the inhibition of enzyme oligomerization, through drug binding to the dimer interface. We have computationally mutated 232 interfacial residues by alanine and calculated the loss in binding free energy among the monomers that build up each dimer of the homotetramer. This led to the identification of the (ten) key residues for the formation of the active dimer (Glu528, Ile531, Met534, Tyr644, Glu665, Asn686, Lys692, Lys735, Met742, and Val863). The results show that these residues are located in two specific spots of the protein with a cleft shape, whose shape and size is favorable for small drug binding. It is expectable that small molecules specifically bound to these druggable pockets will have a major effect on the oligomerization of the protein or/and in active site formation. This paves the way for the discovery of new families of inhibitors of HMG-CoA-R.

Discovery of New Sites for Drug Binding to the Hypertension-Related Renin-Angiotensinogen Complex

Renin (REN) is a key drug target to stop the hypertension cascade, but thus far only one direct inhibitor has been made commercially available. In this study, we assess an innovative REN inhibition strategy, by targeting the interface of the renin:angiotensinogen (REN:ANG) complex. We characterized the energetic role of interfacial residues of REN:ANG and identified the ones responsible for protein:protein binding, which can serve as drug targets for disruption of the REN:ANG association. For this purpose, we applied a computational alanine scanning mutagenesis protocol, which measures the contribution of each side chain for the protein:protein binding free energy with an accuracy of ≈ 1 kcal/mol. As a result, in REN and ANG, six and eight residues were found to be critical for binding, respectively. The leading force behind REN:ANG complexation was found to be the hydrophobic effect. The binding free energy per residue was found to be proportional to the buried area. Residues responsible for binding were occluded from water at the complex, which promotes an efficient pairing between the two proteins. Two druggable pockets involving critical residues for binding were found on the surface of REN, where small druglike molecules can bind and disrupt the ANG:REN association that may provide an efficient way to achieve REN inhibition and control hypertension.