Quality Assessment of Selected Protein Structures Derived from Homology Modeling and AlphaFold.

With technology advancing, many prediction algorithms have been developed to facilitate the modeling of inherently dynamic and flexible macromolecules such as proteins. Improvements in the prediction of protein structures have attracted a great deal of attention due to the advantages they offer, e.g., in drug design. While trusted experimental methods, such as X-ray crystallography, NMR spectroscopy, and electron microscopy, are preferred structure analysis techniques, in silico approaches are also being widely used. Two computational methods, which are on opposite ends of the spectrum with respect to their modus operandi, i.e., homology modeling and AlphaFold, have been established to provide high-quality structures. Here, a comparative study of the quality of structures either predicted by homology modeling or by AlphaFold is presented based on the characteristics determined by experimental studies using structure validation servers to fulfill the purpose. Although AlphaFold is able to predict high-quality structures, high-confidence parts are sometimes observed to be in disagreement with experimental data. On the other hand, while the structures obtained from homology modeling are successful in incorporating all aspects of the experimental structure used as a template, this method may struggle to accurately model a structure in the absence of a suitable template. In general, although both methods produce high-quality models, the criteria by which they are superior to each other are different and thus discussed in detail.

Targeting Gαi/s Proteins with Peptidyl Nucleotide Exchange Modulators.

Chemical probes that specifically modulate the activity of heterotrimeric G proteins provide excellent tools for investigating G protein-mediated cell signaling. Herein, we report a family of selective peptidyl Gαi/s modulators derived from peptide library screening and optimization. Conjugation to a cell-penetrating peptide rendered the peptides cell-permeable and biologically active in cell-based assays. The peptides exhibit potent guanine-nucleotide exchange modulator-like activity toward Gαi and Gαs. Molecular docking and dynamic simulations revealed the molecular basis of the protein-ligand interactions and their effects on GDP binding. This study demonstrates the feasibility of developing direct Gαi/s modulators and provides a novel chemical probe for investigating cell signaling through GPCRs/G proteins.

Strategies towards Targeting Gαi/s Proteins: Scanning of Protein-Protein Interaction Sites To Overcome Inaccessibility.

Heterotrimeric G proteins are classified into four subfamilies and play a key role in signal transduction. They transmit extracellular signals to intracellular effectors subsequent to the activation of G protein-coupled receptors (GPCRs), which are targeted by over 30 % of FDA-approved drugs. However, addressing G proteins as drug targets represents a compelling alternative, for example, when G proteins act independently of the corresponding GPCRs, or in cases of complex multifunctional diseases, when a large number of different GPCRs are involved. In contrast to Gαq, efforts to target Gαi/s by suitable chemical compounds has not been successful so far. Here, a comprehensive analysis was conducted examining the most important interface regions of Gαi/s with its upstream and downstream interaction partners. By assigning the existing compounds and the performed approaches to the respective interfaces, the druggability of the individual interfaces was ranked to provide perspectives for selective targeting of Gαi/s in the future.

Revisiting the interaction of heme with hemopexin.

In hemolytic disorders, erythrocyte lysis results in massive release of hemoglobin and, subsequently, toxic heme. Hemopexin is the major protective factor against heme toxicity in human blood and currently considered for therapeutic use. It has been widely accepted that hemopexin binds heme with extraordinarily high affinity of <1 pM in a 1:1 ratio. However, several lines of evidence point to a higher stoichiometry and lower affinity than determined 50 years ago. Here, we re-analyzed these data. SPR and UV/Vis spectroscopy were used to monitor the interaction of heme with the human protein. The heme-binding sites of hemopexin were characterized using hemopexin-derived peptide models and competitive displacement assays. We obtained a KD value of 0.32 ± 0.04 nM and the ratio for the interaction was determined to be 1:1 at low heme concentrations and at least 2:1 (heme:hemopexin) at high concentrations. We were able to identify two yet unknown potential heme-binding sites on hemopexin. Furthermore, molecular modelling with a newly created homology model of human hemopexin suggested a possible recruiting mechanism by which heme could consecutively bind several histidine residues on its way into the binding pocket. Our findings have direct implications for the potential administration of hemopexin in hemolytic disorders.

NMR-Based Structural Characterization of a Two-Disulfide-Bonded Analogue of the FXIIIa Inhibitor Tridegin: New Insights into Structure-Activity Relationships.

The saliva of blood-sucking leeches contains a plethora of anticoagulant substances. One of these compounds derived from Haementeria ghilianii, the 66mer three-disulfide-bonded peptide tridegin, specifically inhibits the blood coagulation factor FXIIIa. Tridegin represents a potential tool for antithrombotic and thrombolytic therapy. We recently synthesized two-disulfide-bonded tridegin variants, which retained their inhibitory potential. For further lead optimization, however, structure information is required. We thus analyzed the structure of a two-disulfide-bonded tridegin isomer by solution 2D NMR spectroscopy in a combinatory approach with subsequent MD simulations. The isomer was studied using two fragments, i.e., the disulfide-bonded N-terminal (Lys1-Cys37) and the flexible C-terminal part (Arg38-Glu66), which allowed for a simplified, label-free NMR-structure elucidation of the 66mer peptide. The structural information was subsequently used in molecular modeling and docking studies to provide insights into the structure-activity relationships. The present study will prospectively support the development of anticoagulant-therapy-relevant compounds targeting FXIIIa.

Molecular Insights and Functional Consequences of the Interaction of Heme with Activated Protein C.

Aims: In hemolysis, which is accompanied by increased levels of labile redox-active heme and is often associated with hemostatic abnormalities, a decreased activity of activated protein C (APC) is routinely detected. APC is a versatile enzyme that exerts its anticoagulant function through inactivation of clotting factors Va and VIIIa. APC has not been demonstrated to be affected by heme as described for other clotting factors and, thus, is a subject of investigation. Results: We report the interaction of heme with APC and its impact on the protein function by employing spectroscopic and physiologically relevant methods. Binding of heme to APC results in inhibition of its amidolytic and anticoagulant activity, increase of the peroxidase-like activity of heme, and protection of human umbilical vein endothelial cells from heme-induced hyperpermeability. To define the sites that are responsible for heme binding, we mapped the surface of APC for potential heme-binding motifs. T285GWGYHSSR293 and W387IHGHIRDK395, both located on the basic exosite, turned out as potential heme-binding sites. Molecular docking employing a homology model of full-length APC indicated Tyr289 and His391 as the Fe(III)-coordinating amino acids. Innovation: The results strongly suggest that hemolysis-derived heme may directly influence the protein C pathway through binding to APC, conceivably explaining the decreased activity of APC under hemolytic conditions. Further, these results extend our understanding of heme as a multifaceted effector molecule within coagulation and may allow for an improved understanding of disease development in hemostasis under hemolytic conditions. Conclusion: Our study identifies APC as a heme-binding protein and provides insights into the functional consequences.

Distinct 3-disulfide-bonded isomers of tridegin differentially inhibit coagulation factor XIIIa: The influence of structural stability on bioactivity.

Tridegin is a 66mer cysteine-rich coagulation factor XIIIa (FXI-IIa) inhibitor from the giant amazon leech Haementeria ghilianii of yet unknown disulfide connectivity. This study covers the structural and functional characterization of five different 3-disulfide-bonded tridegin isomers. In addition to three previously identified isomers, one isomer containing the inhibitory cystine knot (ICK, knottin) motif, and one isomer with the leech antihemostatic protein (LAP) motif were synthesized in a regioselective manner. A fluorogenic enzyme activity assay revealed a positive correlation between the constriction of conformational flexibility in the N-terminal part of the peptide and the inhibitory potential towards FXI-IIa with clear differences between the isomers. This observation was supported by molecular dynamics (MD) simulations and subsequent molecular docking studies. The presented results provide detailed structure-activity relationship studies of different tridegin disulfide isomers towards FXI-IIa and reveal insights into the possibly existing native linkage compared to non-native disulfide tridegin species.

Phenolic compounds, saponins and alkaloids on cancer progression: emphasis on p53 expression and telomere length.

Telomere length is correlated with cell proliferation, and cancer cells are characterized by an uncontrolled cell cycle. Being apoptosis one of the checks and balances incorporated into cells cycle, due to its characteristics, cancer cells are able to overcome this process. In particular, the tumour suppressor protein p53 loss or inactivation can lead to activation of telomerase enzyme, which can make cells unable to detect DNA damages that spurs apoptosis. Some bioactive compounds, in particular phenolic compounds, saponins and alkaloids have revealed good abilities to affect p53 expression and indirectly control the telomere length. In this sense, this review gives a key emphasis to the ability of these compounds in blocking cancer progression by acting on p53 expression and controlling telomere length. As main findings, phenolic compounds, saponins and alkaloids interfere with cancer progression by stimulating p53 expression, which can cause pro-apoptotic onset and restrict the anti-apoptotic activity, in addition to preventing telomerase enzyme activity.

HeMoQuest: a webserver for qualitative prediction of transient heme binding to protein motifs.

BACKGROUND: The notion of heme as a regulator of many physiological processes via transient binding to proteins is one that is recently being acknowledged. The broad spectrum of the effects of heme makes it important to identify further heme-regulated proteins to understand physiological and pathological processes. Moreover, several proteins were shown to be functionally regulated by interaction with heme, yet, for some of them the heme-binding site(s) remain unknown. The presented application HeMoQuest enables identification and qualitative evaluation of such heme-binding motifs from protein sequences. RESULTS: We present HeMoQuest, an online interface (http://bit.ly/hemoquest) to algorithms that provide the user with two distinct qualitative benefits. First, our implementation rapidly detects transient heme binding to nonapeptide motifs from protein sequences provided as input. Additionally, the potential of each predicted motif to bind heme is qualitatively gauged by assigning binding affinities predicted by an ensemble learning implementation, trained on experimentally determined binding affinity data. Extensive testing of our implementation on both existing and new manually curated datasets reveal that our method produces an unprecedented level of accuracy (92%) in identifying those residues assigned "heme binding" in all of the datasets used. Next, the machine learning implementation for the prediction and qualitative assignment of binding affinities to the predicted motifs achieved 71% accuracy on our data. CONCLUSIONS: Heme plays a crucial role as a regulatory molecule exerting functional consequences via transient binding to surfaces of target proteins. HeMoQuest is designed to address this imperative need for a computational approach that enables rapid detection of heme-binding motifs from protein datasets. While most existing implementations attempt to predict sites of permanent heme binding, this application is to the best of our knowledge, the first of its kind to address the significance of predicting transient heme binding to proteins.

A Computational Approach for Mapping Heme Biology in the Context of Hemolytic Disorders.

Heme is an iron ion-containing molecule found within hemoproteins such as hemoglobin and cytochromes that participates in diverse biological processes. Although excessive heme has been implicated in several diseases including malaria, sepsis, ischemia-reperfusion, and disseminated intravascular coagulation, little is known about its regulatory and signaling functions. Furthermore, the limited understanding of heme's role in regulatory and signaling functions is in part due to the lack of curated pathway resources for heme cell biology. Here, we present two resources aimed to exploit this unexplored information to model heme biology. The first resource is a terminology covering heme-specific terms not yet included in standard controlled vocabularies. Using this terminology, we curated and modeled the second resource, a mechanistic knowledge graph representing the heme's interactome based on a corpus of 46 scientific articles. Finally, we demonstrated the utility of these resources by investigating the role of heme in the Toll-like receptor signaling pathway. Our analysis proposed a series of crosstalk events that could explain the role of heme in activating the TLR4 signaling pathway. In summary, the presented work opens the door to the scientific community for exploring the published knowledge on heme biology.

Structural insights into heme binding to IL-36α proinflammatory cytokine.

Cytokines of the interleukin (IL)-1 family regulate immune and inflammatory responses. The recently discovered IL-36 family members are involved in psoriasis, rheumatoid arthritis, and pulmonary diseases. Here, we show that IL-36α interacts with heme thereby contributing to its regulation. Based on in-depth spectroscopic analyses, we describe two heme-binding sites in IL-36α that associate with heme in a pentacoordinated fashion. Solution NMR analysis reveals structural features of IL-36α and its complex with heme. Structural investigation of a truncated IL-36α supports the notion that the N-terminus is necessary for association with its cognate receptor. Consistent with our structural studies, IL-36-mediated signal transduction was negatively regulated by heme in synovial fibroblast-like synoviocytes from rheumatoid arthritis patients. Taken together, our results provide a structural framework for heme-binding proteins and add IL-1 cytokines to the group of potentially heme-regulated proteins.

Effect of Conformational Diversity on the Bioactivity of µ-Conotoxin PIIIA Disulfide Isomers.

Cyclic µ-conotoxin PIIIA, a potent blocker of skeletal muscle voltage-gated sodium channel NaV1.4, is a 22mer peptide stabilized by three disulfide bonds. Combining electrophysiological measurements with molecular docking and dynamic simulations based on NMR solution structures, we investigated the 15 possible 3-disulfide-bonded isomers of µ-PIIIA to relate their blocking activity at NaV1.4 to their disulfide connectivity. In addition, three µ-PIIIA mutants derived from the native disulfide isomer, in which one of the disulfide bonds was omitted (C4-16, C5-C21, C11-C22), were generated using a targeted protecting group strategy and tested using the aforementioned methods. The 3-disulfide-bonded isomers had a range of different conformational stabilities, with highly unstructured, flexible conformations with low or no channel-blocking activity, while more constrained molecules preserved 30% to 50% of the native isomer's activity. This emphasizes the importance and direct link between correct fold and function. The elimination of one disulfide bond resulted in a significant loss of blocking activity at NaV1.4, highlighting the importance of the 3-disulfide-bonded architecture for µ-PIIIA. µ-PIIIA bioactivity is governed by a subtle interplay between an optimally folded structure resulting from a specific disulfide connectivity and the electrostatic potential of the conformational ensemble.

Coagulation Factor XIIIa Inhibitor Tridegin: On the Role of Disulfide Bonds for Folding, Stability, and Function.

Tridegin is a potent and specific 66mer peptide inhibitor of coagulation factor XIIIa with six cysteines involved in three disulfide bonds. Three of the 15 possible 3-disulfide-bonded isomers have been identified, which share a bridge between cysteines 19 and 25. We synthesized the three possible 2-disulfide-bonded analogues using a targeted protecting group strategy to investigate the impact of the C19-C25 bond on tridegin's folding, stability, and function. The FXIIIa inhibitory activity of the analogues was retained, which was shown by in vitro fluorogenic activity and whole blood clotting assays. Molecular dynamics simulations of wild-type tridegin and the analogues as well as molecular docking studies with FXIIIa were performed to elucidate the impact of the C19-C25 bond on conformational stability and binding mode. The strategy of selectively reducing disulfide bonds to facilitate large-scale synthesis, while retaining the functionality of disulfide-bonded peptides, has been demonstrated with our present study.

The molecular basis of transient heme-protein interactions: analysis, concept and implementation.

Deviant levels of available heme and related molecules can result from pathological situations such as impaired heme biosynthesis or increased hemolysis as a consequence of vascular trauma or bacterial infections. Heme-related biological processes are affected by these situations, and it is essential to fully understand the underlying mechanisms. While heme has long been known as an important prosthetic group of various proteins, its function as a regulatory and signaling molecule is poorly understood. Diseases such as porphyria are caused by impaired heme metabolism, and heme itself might be used as a drug in order to downregulate its own biosynthesis. In addition, heme-driven side effects and symptoms emerging from heme-related pathological conditions are not fully comprehended and thus impede adequate medical treatment. Several heme-regulated proteins have been identified in the past decades, however, the molecular basis of transient heme-protein interactions remains to be explored. Herein, we summarize the results of an in-depth analysis of heme binding to proteins, which revealed specific binding modes and affinities depending on the amino acid sequence. Evaluating the binding behavior of a plethora of heme-peptide complexes resulted in the implementation of a prediction tool (SeqD-HBM) for heme-binding motifs, which eventually led and will perspectively lead to the identification and verification of so far unknown heme-regulated proteins. This systematic approach resulted in a broader picture of the alternative functions of heme as a regulator of proteins. However, knowledge on heme regulation of proteins is still a bottomless barrel that leaves much scope for future research and development.

Insights into the Folding of Disulfide-Rich µ-Conotoxins.

The study of protein conformations using molecular dynamics (MD) simulations has been in place for decades. A major contribution to the structural stability and native conformation of a protein is made by the primary sequence and disulfide bonds formed during the folding process. Here, we investigated µ-conotoxins GIIIA, KIIIA, PIIIA, SIIIA, and SmIIIA as model peptides possessing three disulfide bonds. Their NMR structures were used for MD simulations in a novel approach studying the conformations between the folded and the unfolded states by systematically breaking the distinct disulfide bonds and monitoring the conformational stability of the peptides. As an outcome, the use of a combination of the existing knowledge and results from the simulations to classify the studied peptides within the extreme models of disulfide folding pathways, namely the bovine pancreatic trypsin inhibitor pathway and the hirudin pathway, is demonstrated. Recommendations for the design and synthesis of cysteine-rich peptides with a reduced number of disulfide bonds conclude the study.

Insights into mechanism and functional consequences of heme binding to hemolysin-activating lysine acyltransferase HlyC from Escherichia coli.

BACKGROUND: Tight regulation of heme homeostasis is a critical mechanism in pathogenic bacteria since heme functions as iron source and prosthetic group, but is also toxic at elevated concentrations. Hemolysin-activating lysine-acyltransferase (HlyC) from Escherichia coli is crucial for maturation of hemolysin A, which lyses several mammalian cells including erythrocytes liberating large amounts of heme for bacterial uptake. A possible impact and functional consequences of the released heme on events employing bacterial HlyC have remained unexplored. METHODS: Heme binding to HlyC was investigated using UV/vis and SPR spectroscopy. Functional impact of heme association was examined using an in vitro hemolysis assay. The interaction was further studied by homology modeling, molecular docking and dynamics simulations. RESULTS: We identified HlyC as potential heme-binding protein possessing heme-regulatory motifs. Using wild-type protein and a double alanine mutant we demonstrated that heme binds to HlyC via histidine 151 (H151). We could show further that heme inhibits the enzymatic activity of wild-type HlyC. Computational studies illustrated potential interaction sites in addition to H151 confirming the results from spectroscopy indicating more than one heme-binding site. CONCLUSIONS: Taken together, our results reveal novel insights into heme-protein interactions and regulation of a component of the heme uptake system in one of the major causative agents of urinary tract infections in humans. GENERAL SIGNIFICANCE: This study points to a possible novel mechanism of regulation as present in many uropathogenic E. coli strains at an early stage of heme iron acquisition from erythrocytes for subsequent internalization by the bacterial heme-uptake machinery.

Obstructive Sleep Apnea: Role of an Otorhinolaryngologist.

Obstructive sleep apnea is a disorder resulting from collapse of the upper airway during sleep. Its etiology is multifactorial, resulting from the interdependence of structurally vulnerable upper airway anatomy interacting with physiologic mechanism of ventilator instability during sleep. The ENT causes for OSA are relatively simple conditions that can be treated by safe and simple medical and/or surgical procedures. To assess the prevalence of ENT disorders in patients presenting to the sleep clinic. Patients presented to sleep clinic were submitted to an assessment protocol including clinical history, otorhinolaryngology examination and a polysomnography. Total 69 patients were included and distributed into two groups according to AHI: patients with sleep disordered breathing only (simple snorer and/or AHI ≤ 5) and patients with obstructive sleep apnea syndrome (AHI > 5). There was significant statistical difference for deviated nasal septum (p = 0.0004) and inferior turbinate hypertrophy (p = 0.03) in both groups. Most patients were in the class III and IV of Mallampati classification. Odds of having OSA increases more than 1.5 folds as the level of Mallampati classification increases by one class. ENT disorders were more common in the patients with OSA than in simple snorers and have impact on pathophysiology of OSA and its treatment modality. Hence, ENT examination in all patients with sleep disordered breathing will be helpful.

Lidocine Infiltration Test: An Useful Test in the Prediction of Results of Styloidectomy for Eagle's Syndrome.

Eagle's syndrome is caused by elongated styloid process. Its accepted treatment is styloidectomy. However more than one-fourths of patients undergoing styloidectomy do not experience relief. To find the utility of the lidocaine infiltration test to predict the results of styloidectomy in patients clinically diagnosed as having stylalgia. Twenty-six patients undergoing styloidectomy for Eagle's syndrome were included in the study. They were divided into two groups depending on their response to lidocaine infiltration in the tonsillar fossa. Patients were followed up till 3 months after styloidectomy and their pre operative visual analogue scale for pain was compared with the post operative VAS score. Majority of the patients were females and in the fifth decade of life. There were 18 patients in group I and eight patients in Group II. The groups were similar in terms of age and sex distribution and pre operative VAS score for pain. There was good corelation between post infiltration and post operative VAS scores. The test had 94.44 % sensitivity and 87.5 % specificity. The age and sex distribution and the failure rates in the present study were similar to that reported in other studies. There are many other reasons besides elongation which can cause the typical pain of stylalgia and some of them are not amenable to styloidectomy. The lidocaine infiltration test is an useful test to predict the results of styloidectomy for Eagle's syndrome.

Immune reconstitution disease or mycobacteria other than tuberculosis or both: A dilemma in a patient of AIDS.

A 35-year-old male diagnosed as HIV with tuberculous lymphadenopathy, presented with acute increase in size of neck swelling and fever. The patient was on antiretroviral therapy and antitubercular treatment. Investigations revealed raised CD4 counts and the pus from swelling showed mycobacteria other than tuberculosis (MOTT) on bacteriological examination.The patient was started on steroids, azithromycin, and ciprofloxacin to which he responded well. We report this case to highlight the occurrence of immune reconstitution disease in HIV patients and also to bring out the fact that atypical infection like MOTT may confound the diagnosis even in regions like ours where MOTT is rarely reported.

A randomized control trial to verify the efficacy of pre-operative intra venous tranexamic Acid in the control of tonsillectomy bleeding.

Tonsillectomy is an age old surgery which is still very commonly done. Bleeding related to surgery is the major problem. This study is done to verify by randomized control trial the efficacy of preoperative intravenous tranexamic acid in the control of tonsillectomy bleeding. Hundred cases undergoing tonsillectomy were randomized into two groups, one of which received pre-operatively intra venous tranexamic acid, 10 mg kg(-1). The other group patients were given a placebo. Amount of bleeding was assessed in each case. The study group had statistically highly significant reduction in bleeding. There were no side effects of the drug. This finding is similar to that in other studies for tonsillectomy, other surgeries and other hemorrhagic conditions. Tranexamic acid in the dose of 10 mg kg(-1) given intra venous pre-operatively is effective in the control of tonsillectomy bleeding.