Integrated computational approaches and tools for allosteric drug discovery:

Understanding molecular mechanisms underlying the complexity of allosteric regulation in proteins has attracted considerable attention in drug discovery due to the benefits and versatility of allosteric modulators in providing desirable selectivity against protein targets while minimizing toxicity and other side effects. The proliferation of novel computational approaches for predicting ligand–protein interactions and binding using dynamic and network-centric perspectives has led to new insights into allosteric mechanisms and facilitated computer-based discovery of allosteric drugs. Although no absolute method of experimental and in silico allosteric drug/site discovery exists, current methods are still being improved. As such, the critical analysis and integration of established approaches into robust, reproducible, and customizable computational pipelines with experimental feedback could make allosteric drug discovery more efficient and reliable. In this article, we review computational approaches for allosteric drug discovery and discuss how these tools can be utilized to develop consensus workflows for in silico identification of allosteric sites and modulators with some applications to pathogen resistance and precision medicine. The emerging realization that allosteric modulators can exploit distinct regulatory mechanisms and can provide access to targeted modulation of protein activities could open opportunities for probing biological processes and in silico design of drug combinations with improved therapeutic indices and a broad range of activities

Constrained optimization applied to multiscale integrative modeling

Multiscale integrative modeling stands at the intersection between experimental and computational techniques to predict the atomistic structures of important macromolecules. In the integrative modeling process, the experimental information is often integrated with energy potential and macromolecular substructures in order to derive realistic structural models. This heterogeneous information is often combined into a global objective function that quantifies the quality of the structural models and that is minimized through optimization. In order to balance the contribution of the relative terms concurring to the global function, weight constants are assigned to each term through a computationally demanding process. In order to alleviate this common issue, we suggest to switch from the traditional paradigm of using a single unconstrained global objective function to a constrained optimization scheme. The work presented in this thesis describes the different applications and methods associated with the development of a general constrained optimization protocol for multiscale integrative modeling. The initial implementation concerned the prediction of symmetric macromolecular assemblies throught the incorporation of a recent efficient constrained optimizer nicknamed mViE (memetic Viability Evolution) to our integrative modeling protocol power (parallel optimization workbench to enhance resolution). We tested this new approach through rigorous comparisons against other state-of-the-art integrative modeling methods on a benchmark set of solved symmetric macromolecular assemblies. In this process, we validated the robustness of the constrained optimization method by obtaining native-like structural models. This constrained optimization protocol was then applied to predict the structure of the elusive human Huntingtin protein. Due to the fact that little structural information was available when the project was initiated, we integrated information from secondary structure prediction and low-resolution experiments, in the form of cryo-electron microscopy maps and crosslinking mass spectrometry data, in order to derive a structural model of Huntingtin. The structure resulting from such integrative modeling approach was used to derive dynamic information about Huntingtin protein. At a finer level of resolution, the constrained optimization protocol was then applied to dock small molecules inside the binding site of protein targets. We converted the classical molecular docking problem from an unconstrained single objective optimization to a constrained one by extracting local and global constraints from pre-computed energy grids. The new approach was tested and validated on standard ligand-receptor benchmark sets widely used by the molecular docking community, and showed comparable results to state-of-the-art molecular docking programs. Altogether, the work presented in this thesis proposed improvements in the field of multiscale integrative modeling which are reflected both in the quality of the models returned by the new constrained optimization protocol and in the simpler way of treating the uncorrelated terms concurring to the global scoring scheme to estimate the quality of the models

Structure and Dynamics of Viral Substrate Recognition and Drug Resistance: A Dissertation

Drug resistance is a major problem in quickly evolving diseases, including the human immunodeficiency (HIV) and hepatitis C viral (HCV) infections. The viral proteases (HIV protease and HCV NS3/4A protease) are primary drug targets. At the molecular level, drug resistance reflects a subtle change in the balance of molecular recognition; the drug resistant protease variants are no longer effectively inhibited by the competitive drug molecules but can process the natural substrates with enough efficiency for viral survival. Therefore, the inhibitors that better mimic the natural substrate binding features should result in more robust inhibitors with flat drug resistance profiles. The native substrates adopt a consensus volume when bound to the enzyme, the substrate envelope. The most severe resistance mutations occur at protease residues that are contacted by the inhibitors outside the substrate envelope. To guide the design of robust inhibitors, we investigate the shared and varied properties of substrates with the protein dynamics taken into account to define the dynamic substrate envelope of both viral proteases. The NS3/4A dynamic substrate envelope is compared with inhibitors to detect the structural and dynamic basis of resistance mutation patterns. Comparative analyses of substrates and inhibitors result in a solid list of structural and dynamic features of substrates that are not shared by inhibitors. This study can help guiding the development of novel inhibitors by paying attention to the subtle differences between the binding properties of substrates versus inhibitors

Specificity and evolution of bacterial two-component signal transduction systems

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2013.Cataloged from PDF version of thesis.Includes bibliographical references.Cells possess a remarkable capacity to sense and process a diverse range of signals. Duplication and divergence of a relatively small number of gene families has provided the raw material enabling cells to quickly increase their signaling capacity. After duplication, however, all pathway components are identical in sequence and function. To evolve a new role, the pathways must become insulated at the level of signal transduction. Two-component signal transduction systems, consisting of a sensor histidine kinase and a cognate response regulator, are the main means by which bacteria sense and respond to their environment. These systems have undergone extensive duplication and lateral gene transfer such that most species encode dozens to hundreds of these pathways, yet there is little evidence of cross-talk at the level of signal transduction. Previous work has shown that interaction specificity is dictated by molecular recognition and determined by a small set of specificity residues. I begin by studying the evolutionary trajectories of specificity residues in a duplicated two-component system that lead to insulation of pathways while at the same time maintaining interaction between cognate kinases and regulators. I then examine specificity residues in orthologs of a single two-component system and show that specificity residues are typically under purifying selection, but, as a result of additions to the two-component signaling network, can undergo bursts of diversification followed by extended stasis. By reversing these mutations I demonstrate that avoidance of cross-talk is a major selective pressure. Finally, I show that covalent attachment of the response regulator to a kinase represents an alternative mechanism for enforcing specificity. In these cases, no changes are needed to accommodate a duplication; the high effective concentration of the covalently attached response regulator prevents cross-talk with other two component proteins in the cell. This may allow hybrid kinases to be duplicated or transferred between genomes more easily. This work sheds light on the apparent ease with which two-component systems have expanded to become the dominant signaling system in bacterial genomes and, more generally, how a small number of gene families can be responsible for signal transduction in all organisms.by Emily Jordan Capra.Ph.D

A Computational perspective on the concerted cleavage mechanism of the natural targets of HIV-1 protease.

Doctoral Degree. University of KwaZulu-Natal, Durban.One infectious disease that has had both a profound health and cultural impact on the human race in recent decades is the Acquired Immune Deficiency Syndrome (AIDS) caused by the Human Immunodeficiency Virus (HIV). A major breakthrough in the treatment of HIV-1 was the use of drugs inhibiting specific enzymes necessary for the replication of the virus. Among these enzymes is HIV-1 protease (PR), which is an important degrading enzyme necessary for the proteolytic cleavage of the Gag and Gag-Pol polyproteins, required for the development of mature virion proteins. The mechanism of action of the HIV-1 PR on the proteolysis of these polyproteins has been a subject of research over the past three decades. Most investigations on this subject have been dedicated to exploring the reaction mechanism of HIV-1 PR on its targets as a stepwise general acid-base process with little attention on a concerted model. One of the shortcomings of the stepwise reaction pathway is the existence of more than two TS moieties, which have led to varying opinions on the exact rate-determining step of the reaction and the protonation pattern of the catalytic aspartate group at the HIV-1 PR active site. Also, there is no consensus on the actual recognition mechanism of the natural substrates by the HIV-1 PR. By means of concerted transition state (TS) structural models, the recognition mode and the reaction mechanism of HIV-1 PR with its natural targets were investigated in this present study. The investigation was designed to elucidate the cleavage of natural substrates by HIV-1 PR using the concerted TS model through the application of computational methods to unravel the recognition and reaction process, compute activation parameters and elucidate quantum chemical properties of the system. Quantum mechanics (QM) methods including the density functional theory (DFT) models and Hartree-Fock (HF), molecular mechanics (MM) and hybrid QM/MM were employed to provide better insight in this topic. Based on experience with concerted TS modelling, the six-membered ring TS structure was proposed. Using a small model system and QM methods (DFT and HF), the enzymatic mechanism of HIV-1 PR was studied as a general acid-base model having both catalytic aspartate group participating and water molecule attacking the natural substrate synchronously. The natural substrate scissile bond strength was also investigated via changes of electronic effects. The proposed concerted six-membered ring TS mechanism of the natural substrate within the entire enzyme was studied using hybrid QM/MM; “Our own N-layered Integrated molecular Orbital and molecular Mechanics” (ONIOM) method. This investigation led us to a new perspective in which an acyclic concerted pathway provided a better approach to the subject than the proposed six-membered model. The natural substrate recognition pattern was therefore investigated using the concerted acyclic TS modelling to examine if HIV-1 (South Africa subtype C, C-SA and subtype B) PRs recognize their substrates in the same manner using ONIOM approach. A major outcome in the present investigation is the computational modelling of a new, potentially active, substrate-based inhibitor through the six-membered concerted cyclic TS modelling and a small system. By modelling the entire enzyme—substrate system using a hybrid QM/MM (ONIOM) method, three different pathways were obtained. (1) A concerted acyclic TS structure, (2) a concerted six-membered cyclic TS model and (3) another sixmembered ring TS model involving two water molecules. The activation free energies obtained for the first and the last pathways were in agreement with in vitro HIV-1 PR hydrolysis data. The mechanism that provides marginally the lowest activation barrier involves an acyclic TS model with one water molecule at the HIV-1 PR active site. The outcome of the study provides a plausible theoretical benchmark for the concerted enzymatic mechanism of HIV-1 PRs which could be applied to related homodimeric protease and perhaps other enzymatic processes. Applying the one-step concerted acyclic catalytic mechanism for two HIV-1 PR subtypes, the recognition phenomena of both enzyme and substrate were studied. It was observed that the studied HIV-1 PR subtypes (B and C-SA) recognize and cleave at both scissile and non-scissile regions of the natural substrate sequences and maintaining preferential specificity for the scissile bonds with characteristic lower activation free energies. Future studies on the reaction mechanism of HIV-1 PR and natural substrates should involve the application of advanced computational techniques to provide plausible answers to some unresolved perspectives. Theoretical investigations on the enzymatic mechanism of HIV-1 PR— natural substrate in years to come, would likely involve the application of sophisticated computational techniques aimed at exploring more than the energetics of the system. The possibility of integrated computational algorithms which do not involve partitioning/restraining/constraining/cropped model systems of the enzyme—substrate mechanism would likely surface in future to accurately elucidate the HIV-1 PR catalytic process on natural substrates/ligands

The mechanistic modelling of HIV-1 protease and its natural substrates: a theoretical perspective.

Doctoral Degree. University of KwaZulu-Natal, Durban.An epidemic that has had profound impact on humanity both culturally and health-wise in recent decades is the Acquired immunodeficiency syndrome (AIDS) triggered by the Human immunodeficiency virus (HIV). The developments of drugs, impeding specific enzymes essential for the replication of the HIV-1 virus, has been a breakthrough in the treatment of the virus. These enzymes include the HIV-1 protease (PR), which is a significant degrading enzyme necessary for the proteolytic cleavage of the Gag and Gag-Pol polyproteins, needed for the maturation of viral protein. The catalytic mechanism of the HIV-1 PR of these polyproteins is a major subject of investigation over the past decades. Most research on this topic explores the HIV-1 PR mechanism of action on its target as a stepwise general acid-base mechanism with little or no attention to the concerted process. Among the limitations of the stepwise reaction model is the presence of more than two transition state (TS) steps and this led to different views on the precise rate-determining step of the reaction as well as the protonation state of the catalytic aspartate in the active site of the HIV-1 PR. Likewise, consensus on the exact recognition mechanism of the natural substrates by HIV-1 PR is not forthcoming. The present study investigates the recognition approach and mechanism of reaction of the HIV-1 PR with its natural substrate by a means of computational models. It is intended to explain the cleavage mechanism of the reaction as a concerted process through the application of in-silico techniques. This is achieved by computing the activation energies and elucidating the quantum chemical properties of the enzyme-substrate system. An improved understanding of the mechanism will assist in the design of new HIV-1 PR inhibitors. The molecular dynamics (MD) technique with hybrid quantum mechanics and molecular mechanics (QM/MM) method that includes the density functional theory (DFT) and Amber model were utilized to investigate the concerted hydrolysis process. Based on previous studies in our group involving concerted TS modeling, a six-membered ring TS pathway was first considered. This was achieved by employing a small model system and QM methods (Hartree-Fock and DFT) for the enzymatic mechanism of the HIV-1 PR. A general-acid base (GA/GB) model where both catalytic aspartate (Asp) groups are involved in the mechanism, and the water molecule at the active site attacks the natural substrate synchronously, was utilized. A new perspective arose from the study where an acyclic concerted computational model offered activation energies closer to experiment observations than the six-membered ring model. Hence, the proposed concerted acyclic mechanism of the HIV-1 natural substrate within the entire protease was investigated using both multi-layered QM/MM “Our own N-layered Integrated molecular Orbital and molecular Mechanics” (ONIOM) theory and QM/MM MD umbrella sampling method. A comprehensive review about experimental and theoretical results for the interactions between HIV PR and its natural substrates was presented. An important output in the present study is that the acyclic TS model barrier with one water molecule at the HIV-1 PR active site (DFT study), provides marginally, the most accurate activation energies. Similarly, the computational model demonstrated that optimum recognition specificity of the enzyme depends on structural details of the substrates as well as the number of amino acids in the substrate sequence (minimum P5-P5ʹ required). By modelling the entire enzyme—substrate system using a hybrid ONIOM QM/MM method, it was observed that although both subtype B and C-SA HIV-1 PR recognize and cleave at the scissile and non-scissile regions of the natural substrate sequence, the scissile region has a lower activation free energy. In all cases we found activation free energies that are in good agreement with experimental results. Also, the free energy profiles obtained from the umbrella sampling model were in absolute agreement with experimental in vitro HIV-1 PR hydrolysis data. The outcome of this investigations offers a plausible theoretical yardstick for the concerted enzymatic mechanism of the HIV-1 PRs that is pragmatic to related aspartate proteases and possibly other enzymatic processes. Future studies on the reaction mechanism of HIV-1 PR and its natural substrate should encompass the use of advanced theoretical techniques aimed at exploring more than the energetics of the system. The prospect of integrated computational algorithms that does not involve cropped/partitioning/constraining or restraining model systems of the enzyme—substrate mechanism to accurately elucidate the HIV-1 PR catalytic process on natural substrates/inhibitors will be undertaken in our group. Computational investigations on the enzymatic mechanism of the HIV-1 PR—natural substrate involves fine-tuning the scissile amide bond strength through steric and electronic factors. This may lead to the development of potential substrate-based inhibitors with better potency and reduced toxicity. ISIQEPHU Ubhubhane olube nomthelela omkhulu ebuntwini bobabili ngokwemvelo nangokuqonda kwezempilo emashumini eminyaka amuva nje yi-Acquired immunodeficiency syndrome (AIDS) okubangelwa yi-Human immunodeficiency virus (HIV). Ukuthuthuka kwezidakamizwa, okufaka amandla ama-enzyme athile abalulekile ekuphindaphindweni kwegciwane le-HIV-1, kube yimpumelelo ekwelashweni kwaleli gciwane. La ma-enzyme afaka i-HIV-1 proteinase (PR), okuyi-enzyme ebalulekile eyonakalisayo edingekayo ekuhlanzeni kwe-protein ye-Gag ne-GagPol, edingeka ekuvuthweni kweprotheni yegciwane. Indlela ebusayo ye-HIV-1 PR yalezi zipolyprotein iyinto enkulu ephenywayo emashumini eminyaka edlule. Ucwaningo oluningi ngalesi sihloko luhlola indlela esebenza ngayo ye-HIV-1 PR kulokho okukuhlosile njengenyathelo elisisekelo le-acid-base elisebenzayo ngaphandle kokunaka noma lengenayo inqubo ehlanganisiwe. Phakathi kokukhawulelwa kwemodeli yokusabela esezingeni eliphansi kukhona ubukhona bezinyathelo ezingaphezu kwezimbili zokuguqula isimo (TS) futhi lokhu kuholele ekubukweni okuhlukile esilinganisweni esinqunyiwe sokulinganisa sokuphendula kanye nesimo sokuhlasela sethonya elishukumisayo kulowo osebenzayo indawo ye-HIV-1 PR. Ngokunjalo, ukuvumelana mayelana nendlela ngqo yokuqashelwa kwezakhi zemvelo nge-HIV-1 PR akusondeli. Ucwaningo lwamanje luphenya indlela yokuqashelwa kanye nendlela yokusabela kwe-HIV-1 PR ngesakhiwo sayo esingokwemvelo ngezindlela zamamodeli wokuncintisana. Kuhloswe ukuchaza indlela ye-cleavage yokusabela njengenqubo ekhonjiwe ngokusebenzisa amasu we-in-silico. Lokhu kutholakala ngokuhlanganisa amandla we-activation amandla kanye nokucacisa izakhiwo zamakhemikhali we-quantum wohlelo lwangaphansi lwe-enzyme. Ukuqonda okungcono kwendlela ezokusiza ekwakhiweni kwama-inhibitors amasha we-HIV-1 PR. Indlela esetshenziswayo yama-molecule (i-MD) ene-hybrid quantum mechanics kanye nemolecule mechanics (QM / MM) efaka inqubo yokusizakala yokusebenza kwe-density theory (DFT) kanye ne-Amber model ukuphenya inqubo ekhonjiwe ye-hydrolysis. Ngokusekelwe kwizifundo zangaphambili eqenjini lethu ezibandakanya ukumodelwa kwe-TS ekhonjiwe, indlela eyindilinga eyisithupha yomgwaqo eyi-TS yaqala ukubhekwa. Lokhu kutholwe ngokusebenzisa uhlelo olusha lwemodeli nezindlela ze-QM (Hartree-Fock ne-DFT) ngomshini we-enzymatic we- HIV-1 PR. Imodeli ejwayelekile ye-acid-(GA / GB) lapho amaqembu womabili we-catalytic aspartate (Asp) abandakanyeka khona emshinini, futhi i-molecule lamanzi esakhiweni esisebenzayo lihlasela i-substrate yemvelo ngokuvumelanisa, lalisetshenziswa. Kuqhamuke umbono omusha ocwaningweni lapho imodeli ye-acyclic ekhonjiwe yokuhlinzekwa kwamandla inika amandla okusebenzisa eduze nokuhlolwa okubonwayo kunasekuqaleni kwendandatho eyindandatho eyisithupha. Ngakho-ke, indlela ehlongozwayo ekhonjwe ngendlela ekhanyayo yeHIV-1 substrate yemvelo kuyo yonke iprotease iphenyisisiwe kusetshenziswa ama-QM / MM amaningi ahlukaniswe ngama-Mechanics”(ONIOM) kanye ne-QM / MM MD isampula isambulela indlela. Ukubuyekezwa okuphelele mayelana nemiphumela yokulinga kanye nemibhalo theory yokuxhumana phakathi kwe-HIV PR nezakhi zayo zemvelo kwalethwa. Umphumela obalulekile ocwaningweni lwamanje ukuthi isithintelo se-acyclic TS imodeli nge-mocule eyodwa yamanzi kwisiza esisebenzayo se-HIV-1 PR (i-DFT), sinikela ngamandla, amandla anembe kakhulu okusebenza. Ngokufanayo, imodeli yokuhlanganisa ibonise ukuthi ukuqashelwa okuphelele kweenzyme kuncike kwimininingwane yokwakheka kwama-substrates kanye nenani lama-amino acid ngokulandelana kwe-substrate (ubuncane be-P5-P5'). Ngokumodela yonke i-enzyme — uhlelo olusebenzisa uhlelo lwe-hybrid ONIOM QM / MM, kwaqapheleka ukuthi yize zombili izifunda ezingaphansi kwe-B ne-C-SA ye-HIV-1 PR zibona futhi zinamathele ezindaweni ezibucayi nezingasontekile zendlela yokulandelana engokwemvelo. isifunda esinomswakama sinamandla aphansi we-activation mahhala. Kuzo zonke izimo sithole amandla we-activation mahhala avumelane kahle nemiphumela yokuhlolwa. Futhi, amaphrofayili wamandla wamahhala atholakala kusampuli yesampuli ye-umbrella ayesesivumelwaneni ngokuphelele nedatha yokuhlolwa kwe-vitro HIV-1 PR hydrolysis. Umphumela walolu phenyo uhlinzeka ngokungenaphutha kwethiyori eyingqophamlando ye-enzymatic mechanism ye-HIV-1 PRs edlulele kumaphrotheni ahlobene ne-aspartate kanye nezinye izinqubo ze-enzymatic. Izifundo zesikhathi esizayo mayelana nendlela yokusebenza kwe-HIV-1 PR kanye nengxenye yayo yemvelo kufanele ifake phakathi ukusetshenziswa kwamasu athuthukile we-theorytical okuhloswe ngawo ukuthola ngaphezu komfutho we-system. Ithemba lama-algorithms ahlanganisiwe wokubandakanya okungabandakanyanga okuhlanganisiwe / ukwahlukanisa / ukuphoqelela noma ukuvimba izindlela eziyimodeli ze-enzyme-inqubo engaphansi yokwengeza ukucacisa ngokunembile inqubo yokulwa ne-HIV-1 PR kuzakhi zangaphansi zemvelo / ezinqandweni kuzokwenziwa eqenjini lethu. Uphenyo lwe-computational mayelana ne-enzymatic mechanism ye-HIV-1 PR-substrate yemvelo ifaka phakathi ukulungisa kahle amandla e-bond ayisihlanganisi nge-steric ne-elekthronikhi. Lokhu kungaholela ekwakhiweni kwama-inhibitors angaphansi komhlaba angaphansi nge-potency engcono nokunciphisa ubuthi

Role of aryl hydrocarbon receptors in infection and inflammation

The aryl hydrocarbon receptor (AhR) is a transcription factor that is activated by various ligands, including pollutants, microorganisms, and metabolic substances. It is expressed extensively in pulmonary and intestinal epithelial cells, where it contributes to barrier defense. The expression of AhR is pivotal in regulating the inflammatory response to microorganisms. However, dysregulated AhR expression can result in endocrine disorders, leading to immunotoxicity and potentially promoting the development of carcinoma. This review focuses on the crucial role of the AhR in facilitating and limiting the proliferation of pathogens, specifically in relation to the host cell type and the species of etiological agents involved in microbial pathogen infections. The activation of AhR is enhanced through the IDO1-AhR-IDO1 positive feedback loop, which is manipulated by viruses. AhR primarily promotes the infection of SARS-CoV-2 by inducing the expression of angiotensin-converting enzyme 2 (ACE2) and the secretion of pro-inflammatory cytokines. AhR also plays a significant role in regulating various types of T-cells, including CD4+ T cells and CD8+ T cells, in the context of pulmonary infections. The AhR pathway plays a crucial role in regulating immune responses within the respiratory and intestinal barriers when they are invaded by viruses, bacteria, parasites, and fungi. Additionally, we propose that targeting the agonist and antagonist of AhR signaling pathways could serve as a promising therapeutic approach for combating pathogen infections, especially in light of the growing prevalence of drug resistance to multiple antibiotics

Structure-Guided Engineering of a Multimeric Bacteriophage-Encoded Endolysin PlyC

Emerging antibiotic resistance has become a global health threat. One alternative to antibiotics is bacteriophage-encoded endolysins. Endolysins are peptidoglycan hydrolases produced at the end of the bacteriophage replication cycle resulting in bacterial cell lysis and progeny bacteriophage release. Endolysins are also capable of destroying the Gram-positive bacterial peptidoglycan when applied externally as recombinant proteins. These enzymes typically consist of an enzymatically active domain (EAD) and a separate cell wall binding domain (CBD). Studies have shown therapeutic efficacy of endolysins in vitro and in vivo, with no resistance developed to date. An endolysin from the streptococcal C1 phage, known as PlyC, has the highest activity of any endolysin reported. It also has a unique multimeric structure consisting of one activity subunit (PlyCA) harboring two synergistically acting catalytic domains, GyH and CHAP, and eight identical binding subunits (PlyCB) forming an octameric ring. Groups A, C, and E streptococci as well as Streptococcus uberis are sensitive to the lytic activities of PlyC. In order to harness the potent activity of PlyC for use against other bacteria, we sought to change/extend the host range of PlyC by engineering PlyCB and PlyCA, respectively. We first used a structure-guided mutagenesis method to obtain the single PlyCB monomer subunit, PlyCBK40A E43A (PlyCBm), aiming to study the binding mechanism of PlyCB. Via fluorescence microscopy and binding assays, we determined that PlyCBm retained the host range of the octamer with a much lower binding affinity, which suggests the PlyCB octamer binds concurrently to a specific epitope on the bacterial surface resulting in a tight, stable interaction. Thus, it is not feasible to change/extend the PlyC host range via engineering PlyCB. Next, we proposed a novel design to engineer PlyCA. We successfully created two chimeric endolysins, ClyX-1 and ClyX-2, possessing the synergistic activity of the GyH and CHAP catalytic domains, but extended the host range to include, Streptococcus pneumoniae, Group B streptococci, Streptococcus mutans, and Enterococcus faecalis, all strains previously insensitive to PlyC. Finally, we tested a novel hypothesis that a positively charged catalytic domain could display lytic activity in a CBD-independent manner resulting in a broad host range. Using the PlyC CHAP domain as a model, we converted the net surface charge of the CHAP domain from negative three to positive one through positive seven. Notwithstanding the range of charges, our mutant CHAP domains did not show lytic activity in a CBD-independent manner, suggesting that other factors, like surface charge distribution, need to be considered in such a way of engineering