Molecular cloning and functional characterization of peroxiredoxin 4 (prx 4) in freshwater crayfish, Procambarus clarkii.

Peroxiredoxin (Prx), which is a newly discovered member of the antioxidant protein family, performs important biological functions in intracellular signal transduction. In the present study, a peroxiredoxin 4 gene was cloned from crayfish for the first time and named Pc-prx 4. According to the amino acid sequence signature, Pc-Prx 4 was identified as the typical 2-Cys Prx molecule, which possessed two conserved cysteines (Cys98 and Cys219). Time-course expression patterns post V. harveyi infection revealed that Pc-prx 4 was likely related to crayfish innate immune defense responses. In particular, the highest fold upregulation of the Pc-prx 4 mRNA transcript reached approximately 170 post V. harveyi infection in the crayfish hepatopancreas. The results of the mixed functional oxidase assay showed that rPc-Prx 4△ could resist the damaging effect of reactive oxygen species generated from the thiol/Fe3+/O2- reaction system to some extent. In addition, the results of the RNAi assay revealed that the crayfish survival rate was obviously increased post injection of V. harveyi when Pc-prx 4 was knocked down. Further study revealed that both hemolymph melanization and PO activity were strengthened to different degrees in the RNAi assay. Therefore, we speculated that the increase in the crayfish survival rate was likely due to the increase in hemolymph melanization. The obviously reinforced hemolymph melanization was directly caused by the upregulation of hemolymph PO activity, which was induced by the knockdown of Pc-prx 4. However, further studies are still indispensable for illuminating the molecular mechanism of Pc-prx 4 in the crayfish innate immune defense system.

Enzymatic Assembly of DNA Nanostructures and Fragments with Sequence Overlaps.

Homologous recombination, an evolutionarily conserved DNA double-strand break repair pathway to protect genome stability, has long been exploited for the in vivo and in vitro assembly of multiple DNA duplex fragments in molecular cloning. Whether such methods can also be applied in the self-assembly of DNA nanostructures remains underexplored. Here, we report an enzymatic approach for the self-assembly of high-order DNA constructs with overlapping segments. In our system, a DNA polymerase with exonuclease activity was introduced to produce ssDNA overhangs for specific sticky end cohesion, and as many as 25 DNA structural units were designed to be hierarchically assembled. Using this approach, we successfully constructed a variety of high-order DNA nanostructures, including tubes and extended oligomers, from homogeneous assembly and custom multimers from heterogeneous assembly. Our strategy expands the construction toolbox of complex DNA nanostructures and highlights the potential to enhance the assembly of duplex fragments in molecular cloning.

TCR sequencing and cloning methods for repertoire analysis and isolation of tumor-reactive TCRs.

T cell receptor (TCR) technologies, including repertoire analyses and T cell engineering, are increasingly important in the clinical management of cellular immunity in cancer, transplantation, and other immune diseases. However, sensitive and reliable methods for repertoire analyses and TCR cloning are still lacking. Here, we report on SEQTR, a high-throughput approach to analyze human and mouse repertoires that is more sensitive, reproducible, and accurate as compared with commonly used assays, and thus more reliably captures the complexity of blood and tumor TCR repertoires. We also present a TCR cloning strategy to specifically amplify TCRs from T cell populations. Positioned downstream of single-cell or bulk TCR sequencing, it allows time- and cost-effective discovery, cloning, screening, and engineering of tumor-specific TCRs. Together, these methods will accelerate TCR repertoire analyses in discovery, translational, and clinical settings and permit fast TCR engineering for cellular therapies.

[Cloning of adipor1 and adipor2 genes in Rana dybowskii and its expression pattern upon infection].

Adiponectin receptor 1 (AdipoR1) and Adiponectin receptor 2 (AdipoR2) can bind to adiponectin (AdipoQ) secreted by adipose tissue to participate in various physiological functions of the body. In order to explore the role of AdipoR1 and AdipoR2 in amphibians infected by Aeromonas hydrophila (Ah), the genes adipor1 and adipor2 of Rana dybowskii were cloned by reverse transcription-polymerase chain reaction (RT-PCR) and analyzed by bioinformatics. The tissue expression difference of adipor1 and adipor2 was analyzed by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR), and an inflammatory model of R. dybowskii infected by Ah was constructed. The histopathological changes were observed by hematoxylin-eosin staining (HE staining); the expression profiles of adipor1 and adipor2 after infection were dynamically detected by qRT-PCR and Western blotting. The results show that AdipoR1 and AdipoR2 are cell membrane proteins with seven transmembrane domains. Phylogenetic tree also shows that AdipoR1 and AdipoR2 cluster with the amphibians in the same branch. qRT-PCR and Western blotting results show that adipor1 and adipor2 were up-regulated at different levels of transcription and translation upon Ah infection, but the response time and level were different. It is speculated that AdipoR1 and AdipoR2 participate in the process of bacterial immune response, providing a basis for further exploring the biological functions of AdipoR1 and AdipoR2 in amphibians.

[Cloning, identification and functional analysis of the goat transcription factor c-fos].

C-fos is a transcription factor that plays an important role in cell proliferation, differentiation and tumor formation. The aim of this study was to clone the goat c-fos gene, clarify its biological characteristics, and further reveal its regulatory role in the differentiation of goat subcutaneous adipocytes. We cloned the c-fos gene from subcutaneous adipose tissue of Jianzhou big-eared goats by reverse transcription-polymerase chain reaction (RT-PCR) and analyzed its biological characteristics. Using real-time quantitative PCR (qPCR), we detected the expression of c-fos gene in the heart, liver, spleen, lung, kidney, subcutaneous fat, longissimus dorsi and subcutaneous adipocytes of goat upon induced differentiation for 0 h to 120 h. The goat overexpression vector pEGFP-c-fos was constructed and transfected into the subcutaneous preadipocytes for induced differentiation. The morphological changes of lipid droplet accumulation were observed by oil red O staining and bodipy staining. Furthermore, qPCR was used to test the relative mRNA level of the c-fos overexpression on adipogenic differentiation marker genes. The results showed that the cloned goat c-fos gene was 1 477 bp in length, in which the coding sequence was 1 143 bp, encoding a protein of 380 amino acids. Protein structure analysis showed that goat FOS protein has a basic leucine zipper structure, and subcellular localization prediction suggested that it was mainly distributed in the nucleus. The relative expression level of c-fos was higher in the subcutaneous adipose tissue of goats (P < 0.05), and the expression level of c-fos was significantly increased upon induced differentiation of subcutaneous preadipocyte for 48 h (P < 0.01). Overexpression of c-fos significantly inhibited the lipid droplets formation in goat subcutaneous adipocytes, significantly decreased the relative expression levels of the AP2 and C/EBPß lipogenic marker genes (P < 0.01). Moreover, AP2 and C/EBPß promoter are predicted to have multiple binding sites. In conclusion, the results indicated that c-fos gene was a negative regulatory factor of subcutaneous adipocyte differentiation in goats, and it might regulate the expression of AP2 and C/EBPß gene expression.

[Cloning and expression profile of ZFP36L1 gene in goat].

The purpose of this study was to clone and characterize the ZFP36L1 (zinc finger protein 36-like 1) gene, clarify its expression characteristics, and elucidate its expression patterns in different tissues of goats. Samples of 15 tissues from Jianzhou big-eared goats, including heart, liver, spleen, lung and kidney were collected. Goat ZFP36L1 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR), then the gene and protein sequence were analyzed by online tools. Quantitative real-time polymerase chain reaction (qPCR) was used to detect the expression level of ZFP36L1 in intramuscular preadipocytes in different tissues and adipocytes of goat at different differentiation stages. The results showed that the length of ZFR36L1 gene was 1 224 bp, and the coding sequence (CDS) region was 1 017 bp, encoding 338 amino acids, which was a non-secretory unstable protein mainly located in nucleus and cytoplasm. Tissue expression profile showed that ZFP36L1 gene was expressed in all selected tissues. In visceral tissues, the small intestine showed the highest expression level (P < 0.01). In muscle tissue, the highest expression level was presented in longissimus dorsi muscle (P < 0.01), whereas the expression level in subcutaneous adipose tissue was significantly higher than that in other tissues (P < 0.01). The results of induced differentiation showed that the expression of this gene was up-regulated during adipogenic differentiation of intramuscular precursor adipocytes (P < 0.01). These data may help to clarify the biological function of the ZFP36L1 gene in goat.

Cell activation-based screening of natively paired human T cell receptor repertoires.

Adoptive immune therapies based on the transfer of antigen-specific T cells have been used successfully to treat various cancers and viral infections, but improved techniques are needed to identify optimally protective human T cell receptors (TCRs). Here we present a high-throughput approach to the identification of natively paired human TCRα and TCRß (TCRα:ß) genes encoding heterodimeric TCRs that recognize specific peptide antigens bound to major histocompatibility complex molecules (pMHCs). We first captured and cloned TCRα:ß genes from individual cells, ensuring fidelity using a suppression PCR. We then screened TCRα:ß libraries expressed in an immortalized cell line using peptide-pulsed antigen-presenting cells and sequenced activated clones to identify the cognate TCRs. Our results validated an experimental pipeline that allows large-scale repertoire datasets to be annotated with functional specificity information, facilitating the discovery of therapeutically relevant TCRs.

Cloning, expression, purification and biochemical characterization of the recombinant chitinase enzyme encoded by CTS2 in the budding yeast.

Chitin is a polymer of ß-1,4-linked N-acetylglucosamine (GlcNAc) and plays a central role in the assembly of the fungal cell wall. Chitinases are hydrolytic enzymes that break down glycosidic bonds in the chitin. Chitinases are classified into three categories, endochitinases, exochitinases and N-acetylglucosaminases, according to the manner in which the enzyme cleaves the chitin polymer. Saccharomyces cerevisiae has two chitinase-encoding genes, CTS1 and CTS2. However, whether Cts2p shows a chitinase activity remains unknown. In this study, we have cloned, expressed and purified the recombinant Cts2p protein from bacterial cells. We have demonstrated that Cts2p has a higher chitobiosidase (exochitinase) activity than endochitinase activity, but no N-acetylglucosaminase activity. The optimal temperature for the chitobiosidase activity of Cts2p is 37 °C.

Molecular Cloning and Characterization of WRKY12, A Pathogen Induced WRKY Transcription Factor from Akebia trifoliata.

WRKY transcription factors (TFs), which are plant-specific TFs, play significant roles in plant defense. Here, a pathogen-induced WRKY gene, named AktWRKY12, which was the homologous gene of AtWRKY12, was isolated from Akebia trifoliata. The AktWRKY12 gene has a total length of 645 nucleotides and an open reading frame (ORF) encoding 214 amino acid polypeptides. The characterizations of AktWRKY12 were subsequently performed with the ExPASy online tool Compute pI/Mw, PSIPRED and SWISS-MODEL softwares. The AktWRKY12 could be classified as a member of WRKY group II-c TFs based on sequence alignment and phylogenetic analysis. The results of tissue-specific expression analysis revealed that the AktWRKY12 gene was expressed in all the tested tissues, and the highest expression level was detected in A. trifoliata leaves. Subcellular localization analysis showed that AktWRKY12 was a nuclear protein. Results showed that the expression level of AktWRKY12 significantly increased in A. trifoliata leaves with pathogen infection. Furthermore, heterologous over-expression of AktWRKY12 in tobacco resulted in suppressed expression of lignin synthesis key enzyme genes. Based on our results, we speculate that AktWRKY12 might play a negative role in A. trifoliata responding to biotic stress by regulating the expression of lignin synthesis key enzyme genes during pathogen infection.

CRISPR/Cas9-Mediated Gene Knockout in Cells and Tissues Using Lentivirus.

CRISPR-Cas9 has become a powerful and popular gene editing tool. However, successful application of this tool in the lab can still be quite daunting to many newcomers to molecular biology, mostly because it is a relatively lengthy process involving multiple steps with variations of each step. Here, we provide a reliable, stepwise, and newcomer-friendly protocol to knock out a target gene in wild-type human fibroblasts. This protocol involves sgRNA design using CRISPOR, construction of an "all-in-one" vector expressing both sgRNA and Cas9 using Golden Gate cloning, streamlined production of high-titer lentiviruses in 1 week after molecular cloning, and transduction of cells to generate a knockout cell pool. We further introduce a protocol for lentiviral transduction of ex vivo mouse embryonic salivary epithelial explants. In summary, our protocol is useful for new researchers to apply CRISPR-Cas9 to generate stable gene knockout cells and tissue explants using lentivirus. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: sgRNA design Basic Protocol 2: Cloning sgRNA in plasmid vector containing Cas9 encoding sequence using golden gate cloning Basic Protocol 3: Lentivirus packaging Basic Protocol 4: Lentivirus transduction of cells Basic Protocol 5: Lentivirus transduction of salivary gland epithelial buds.

Molecular cloning and in silico analysis of chalcone isomerase from Polygonum minus.

BACKGROUND: Chalcone isomerase (CHI; EC 5.5.1.6) is one of the key enzymes in the flavonoid biosynthetic pathway that is responsible for the intramolecular cyclization of chalcones into specific 2S-flavanones. METHODS AND RESULTS: In this study, the open reading frame (ORF) of CHI was successfully isolated from the cDNA of Polygonum minus at 711-bp long, encoding for 236 amino acid residues, with a predicted molecular weight of 25.4 kDa. Multiple sequence alignment and phylogenetic analysis revealed that the conserved residues (Thr50, Tyr108, Asn115, and Ser192) in the cleft of CHI enzyme group active site are present in PmCHI protein sequence and classified as type I. PmCHI comprises more hydrophobic residues without a signal peptide and transmembrane helices. The three-dimensional (3D) structure of PmCHI predicted through homology modeling was validated by Ramachandran plot and Verify3D, with values within the acceptable range of a good model. PmCHI was cloned into pET-28b(+) plasmid, expressed in Escherichia coli BL21(DE3) at 16 °C and partially purified. CONCLUSION: These findings contribute to a deeper understanding of the PmCHI protein and its potential for further characterization of its functional properties in the flavonoid biosynthetic pathway.

[Prediction of epitope region and preparation of mouse polyclonal antibody of human Shisa-like protein 1(SHISAL1)].

Objective To investigate antigen optimization of Shisa like protein 1 (SHISAL1) for preparing mouse anti-human SHISAL1 polyclonal antibody and to identify the specificity of the prepared antibody. Methods Bioinformatics was employed to predict the antigenic epitope region of SHISAL1 protein, and then a polypeptide composed of amino acid residues from the site of 28 to 97 of SHISAL1, termed SHISAL1-N, was selected as the antigen. The coding region of SHISAL1-N was cloned by molecular cloning technique, and then it was inserted into pET-28a to generate pET28a-SHISAL1-N recombinant plasmid. The two recombinant plasmids pET28a-SHISAL1-N and pET28a-SHISAL1 were transformed into BL21 (DE3) bacteria and induced to express by IPTG. The two proteins were purified and immunized to female Kunming mice, respectively. The specificities and sensitivities of the acquired antibodies were detected by Western blot analysis, immunoprecipitation and immunofluorescent cytochemical staining. Results pET28a-SHISAL1-N recombinant plasmid was successfully constructed, and the two fused proteins, SHISAL1 and SHISAL1-N, were induced to express. Moreover, two types of SHISAL1 mouse polyclonal antibodies, derived from SHISAL1-N and SHISAL1 antigens, were obtained. Western blot results showed that the antibody prepared from SHISAL1 antigen was less specific and sensitive compared with the antibody prepared from SHISAL1-N antigen which could specifically identify different endogenous SHISAL1 protein. Immunoprecipitation results showed that SHISAL1-N antibody could specifically pull down SHIISAL1 protein in hepatocellular carcinoma cells and immunofluorescence results demonstrated that SHISAL1-N antibody could specifically bind to SHISAL1 protein in the cytoplasm. Conclusion We have optimized the SHISAL1 antigen and prepared the mouse anti-human SHISAL1 polyclonal antibodies successfully, which can be used for Western blot analysis, immunoprecipitation and immunofluorescence cytochemical staining.

A Modular Cloning Toolkit Including CRISPRi for the Engineering of the Human Fungal Pathogen and Biotechnology Host Candida glabrata.

The yeast Candida glabrata is an emerging, often drug-resistant opportunistic human pathogen that can cause severe systemic infections in immunocompromised individuals. At the same time, it is a valuable biotechnology host that naturally accumulates high levels of pyruvate─a valuable chemical precursor. Tools for the facile engineering of this yeast could greatly accelerate studies on its pathogenicity and its optimization for biotechnology. While a few tools for plasmid-based expression and genome engineering have been developed, there is no well-characterized cloning toolkit that would allow the modular assembly of pathways or genetic circuits. Here, by characterizing the Saccharomyces cerevisiae-based yeast molecular cloning toolkit (YTK) in C. glabrata and by adding missing components, we build a well-characterized CgTK (C. glabrata toolkit). We used the CgTK to build a CRISPR interference system for C. glabrata that can be used to generate selectable phenotypes via single-gRNA targeting such as is required for genome-wide library screens.

Cloning, expression and in vitro validation of chimeric multi epitope vaccine candidate against visceral leishmaniasis infection.

Visceral Leishmaniasis or Kala-Azar is one of the most severe and deadly neglected tropical disease caused by the Leishmania parasite. A few number of vaccines are going through different phases in clinical trial but failing of these vaccines in successive phase trial or less efficacy, urge to develop highly immunogenic and cost-effective treatment to get rid of deadly VL. This study focuses on the development of more potent vaccine candidate against VL. The recombinant vaccine candidate LeiSp was expressed in Pichia pastoris, followed by purification and characterization. The purified protein was also tested for any post-translation modification, which favors being a potent immunogenic candidate. Further, the expression modulation of different pro-inflammatory and anti-inflammatory cytokines was evaluated in THP1 cell lines. A significant upregulation in the expression of pro-inflammatory cytokines while no significant changes were observed in the expression of anti-inflammatory cytokines. The impact of recombinant vaccine protein candidates in infected conditions were determined. Here, upon treatment with chimeric vaccine protein candidate, we observed a considerable recovery in the expression level of pro-inflammatory cytokines, which were downregulated upon infection alone. In addition to this, we found a significant decrease in the expression of anti-inflammatory cytokines, which were upregulated during infection alone. We further validated our findings in infected hPBMCs and observed similar expression modulation of pro-inflammatory and anti-inflammatory cytokines with and without treatment. Thus, the present study indicates that the chimeric LeiSp protein which was designed using bioinformatics approaches shows a potential inductive efficacy for pro-inflammatory cytokines in Leishmania-infected cells.

Molecular cloning and functional analysis of polymeric immunoglobulin receptor, pIgR, gene in mandarin fish Siniperca chuatsi.

Polymeric immunoglobulin receptor (pIgR) can bind and transport immunoglobulins (Igs), thus playing a role in mucosal immunity. In this study, pIgR gene was cloned in mandarin fish, Siniperca chuatsi, with the open reading frame (ORF) of 1011 bp, encoding 336 amino acids. The pIgR protein consists of a signal peptide, an extracellular domain, a transmembrane domain and an intracellular region, with the presence of two Ig-like domains (ILDs) in the extracellular domain, as reported in other species of fish. The pIgR gene was expressed in all organs/tissues of healthy mandarin fish, with higher level observed in liver and spleen. Following the immersion infection of Flavobacterium columnare, pIgR transcripts were detected in immune related, especially mucosal tissues, with significantly increased transcription during the first two days of infection. Through transfection of plasmids expressing pIgR, IgT and IgM, pIgR was found to be interacted with IgT and IgM as revealed by co-immunoprecipitation and immunofluorescence.

Cloning and Recombinant Protein Expression in Lactococcus lactis.

The Lactococcus lactis, a Gram-positive bacteria, is an ideal expression host for the overproduction of heterologous proteins in a properly folded and functional form. L. lactis has been identified as an efficient cell factory, generally recognized as safe (GRAS), has a long history of safe use in food production, and is known to have probiotic properties. Key desirable features of L. lactis include the following: (1) rapid growth to high cell densities, not requiring aeration which facilitates large-scale fermentation; (2) its Gram-positive nature precludes the presence of contaminating endotoxins; (3) the capacity to secrete stable recombinant protein into the growth medium with few proteases resulting in a properly folded, full-length protein; and (4) the availability of diverse expression vectors facilitating various cloning options. We have previously described production of several recombinant proteins with varying degrees of predicted structural complexities using the L. lactis pH-dependent P170 promoter. The purpose of this chapter is to provide a detailed protocol for facilitating wider application of L. lactis as a reliable platform for expression of heterologous recombinant proteins in soluble form. Here, we present details of the various steps involved such as cloning of the target gene in appropriate expression plasmid vector, determination of the expression levels of the heterologous protein, and initial purification of the expressed soluble recombinant protein of interest.

Cloning, Expression Analysis and SNP Screening of the kiss1 Gene in Male Schizothorax biddulphi.

Schizothorax biddulphi is an endemic fish distributed only in southern Xinjiang, China. Due to overfishing, water conservancy facilities, and other factors, as well as inherent biological limitations, resource recovery is quite difficult. For endangered fish with slow growth, late sexual maturity, and insufficient natural population supplementation, large-scale artificial reproduction and breeding are important for restoring resources. Therefore, it is urgent to optimize the reproductive regulation methods of the fish. The kiss1 gene is a key regulator of the reproductive regulation cascade, and identifying and analyzing the role of kiss1 are important for further elucidating the reproductive mechanism of S. biddulphi. To understand the characteristics of the kiss1 of S. biddulphi, the full-length cDNA sequence of kiss1 was obtained in this study, and its tissue expression specificity and association with phenotypic traits were analyzed in male fish. The full-length cDNA sequence of kiss1 in S. biddulphi was 658 bp, with an ORF of 327 bp, and encoded a 108-amino acid, unstable protein. Homology results indicated that kiss1 was highly conserved. qPCR showed kiss1 expression in different tissues in male S. biddulphi, with the highest expression in the gonads, followed by muscle, and significantly lower expression in the swim bladder, pituitary gland, heart, hypothalamus, gill, fin, liver, eye, and mid-kidney. qPCR revealed three SNP loci in the exonic region of kiss1. The c.3G>T locus was significantly correlated (p < 0.05) with gonad mass and the maturation coefficient in S. biddulphi. These results will help uncover the reproductive endocrinology network of S. biddulphi, improve artificial breeding technology for fish, and unveil new directions for breeding excellent strains of S. biddulphi and molecular marker-assisted breeding.

Cloning a profibrotic stem cell variant in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible, and rapidly fatal interstitial lung disease marked by the replacement of lung alveoli with dense fibrotic matrices. Although the mechanisms initiating IPF remain unclear, rare and common alleles of genes expressed in lung epithelia, combined with aging, contribute to the risk for this condition. Consistently, single-cell RNA sequencing (scRNA-seq) studies have identified lung basal cell heterogeneity in IPF that might be pathogenic. We used single-cell cloning technologies to generate "libraries" of basal stem cells from the distal lungs of 16 patients with IPF and 10 controls. We identified a major stem cell variant that was distinguished from normal stem cells by its ability to transform normal lung fibroblasts into pathogenic myofibroblasts in vitro and to activate and recruit myofibroblasts in clonal xenografts. This profibrotic stem cell variant, which was shown to preexist in low quantities in normal and even fetal lungs, expressed a broad network of genes implicated in organ fibrosis and showed overlap in gene expression with abnormal epithelial signatures identified in previously published scRNA-seq studies of IPF. Drug screens highlighted specific vulnerabilities of this profibrotic variant to inhibitors of epidermal growth factor and mammalian target of rapamycin signaling as prospective therapeutic targets. This profibrotic stem cell variant in IPF was distinct from recently identified profibrotic stem cell variants in chronic obstructive pulmonary disease and may extend the notion that inappropriate accrual of minor and preexisting stem cell variants contributes to chronic lung conditions.

Cloning and expression of the mitochondrial cytochrome c oxidase subunit II gene in Sitophilus zeamais and interaction mechanism with allyl isothiocyanate.

In the United States, allyl isothiocyanate (AITC) has been registered as an insecticide, bactericide, and nematicide. And it has been confirmed that AITC has significant insecticidal activities against four stored product pests including Sitophilus zeamais Mostchulky (Coleoptera: Curculionidae). This study aimed to verify the mechanism of action of AITC on cytochrome c oxidase core subunits II in S. zeamais. Enzyme - catalyzed reactions and Fourier transform infrared spectrometer (FTIR) analysis revealed that the expressed COX II proteins could competitively bind and inhibit the activity of COX II. Furthermore, molecular docking results showed that a sulfur atom of AITC could form a 2.9 Å hydrogen bond with Ile-30, having a binding energy of -2.46 kcal/mol.