Designing peptides predicted to bind to the omicron variant better than ACE2 via computational protein design and molecular dynamics.

Brought about by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), coronavirus disease (COVID-19) pandemic has resulted in large numbers of worldwide deaths and cases. Several SARS-CoV-2 variants have evolved, and Omicron (B.1.1.529) was one of the important variants of concern. It gets inside human cells by using its S1 subunit's receptor-binding domain (SARS-CoV-2-RBD) to bind to Angiotensin-converting enzyme 2 receptor's peptidase domain (ACE2-PD). Using peptides to inhibit binding interactions (BIs) between ACE2-PD and SARS-CoV-2-RBD is one of promising COVID-19 therapies. Employing computational protein design (CPD) as well as molecular dynamics (MD), this study used ACE2-PD's α1 helix to generate novel 25-mer peptide binders (SPB25) of Omicron RBD that have predicted binding affinities (ΔGbind (MM­GBSA)) better than ACE2 by increasing favorable BIs between SPB25 and the conserved residues of RBD. Results from MD and the MM-GBSA method identified two best designed peptides (SPB25T7L/K11A and SPB25T7L/K11L with ΔGbind (MM­GBSA) of -92.4 ± 0.4 and -95.7 ± 0.5 kcal/mol, respectively) that have better ΔGbind (MM­GBSA) to Omicron RBD than ACE2 (-87.9 ± 0.5 kcal/mol) and SPB25 (-71.6 ± 0.5 kcal/mol). Additionally, they were predicted to have slightly higher stabilities, based on their percent helicities in water, than SBP1 (the experimentally proven inhibitor of SARS-CoV-2-RBD). Our two best designed SPB25s are promising candidates as omicron variant inhibitors.

Computational redesign of Beta-27 Fab with substantially better predicted binding affinity to the SARS-CoV-2 Omicron variant than human ACE2 receptor.

During the COVID-19 pandemic, SARS-CoV-2 has caused large numbers of morbidity and mortality, and the Omicron variant (B.1.1.529) was an important variant of concern. To enter human cells, the receptor-binding domain (RBD) of the S1 subunit of SARS-CoV-2 (SARS-CoV-2-RBD) binds to the peptidase domain (PD) of Angiotensin-converting enzyme 2 (ACE2) receptor. Disrupting the binding interactions between SARS-CoV-2-RBD and ACE2-PD using neutralizing antibodies is an effective COVID-19 therapeutic solution. Previous study found that Beta-27 Fab, which was obtained by digesting the full IgG antibodies that were isolated from a patient infected with SARS-CoV-2 Beta variant, can neutralize Victoria, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2) variants. This study employed computational protein design and molecular dynamics (MD) to investigate and enhance the binding affinity of Beta-27 Fab to SARS-CoV-2-RBD Omicron variant. MD results show that five best designed Beta-27 Fabs (Beta-27-D01 Fab, Beta-27-D03 Fab, Beta-27-D06 Fab, Beta-27-D09 Fab and Beta-27-D10 Fab) were predicted to bind to Omicron RBD in the area, where ACE2 binds, with significantly better binding affinities than Beta-27 Fab and ACE2. Their enhanced binding affinities are mostly caused by increased binding interactions of CDR L2 and L3. They are promising candidates that could potentially be employed to disrupt the binding between ACE2 and Omicron RBD.

Biochemical and ligand binding properties of recombinant Xenopus laevis cortical granule lectin-1.

Intelectins are putative innate immune lectins that are found throughout chordates. The first intelectin reported was Xenopus laevis cortical granule lectin-1 (XCGL-1 or XL-35). XCGL-1 is critical in fertilization membrane development in Xenopus. Here, we explored the biochemical properties of XCGL-1. The cysteines responsible for forming intermolecular disulfide bonds were identified. XCGL-1 adopted a four-lobed structure as observed by electron microscopy. The full-length XCGL-1 and the carbohydrate recognition domain (CRD) bind galactose-containing carbohydrates at nanomolar to micromolar affinities. Molecular modeling suggested that galactoside ligands coordinated the binding site calcium ion and interacted with residues around the groove made available by the non-conserved substitution compared to human intelectin-1. Folding conditions for production of recombinant XCGL-1 CRD were also investigated. Our results not only provide new biochemical insights into the function of XCGL-1, but may also provide foundation for further applications of XCGL-1 as glycobiology tools.

Computational design of Lactobacillus Acidophilus α-L-rhamnosidase to increase its structural stability.

α-L-rhamnosidase catalyzes hydrolysis of the terminal α-L-rhamnose from various natural rhamnoglycosides, including naringin and hesperidin, and has various applications such as debittering of citrus juices in the food industry and flavonoid derhamnosylation in the pharmaceutical industry. However, its activity is lost at high temperatures, limiting its usage. To improve Lactobacillus acidophilus α-L-rhamnosidase stability, we employed molecular dynamics (MD) to identify a highly flexible region, as evaluated by its root mean square fluctuation (RMSF) value, and computational protein design (Rosetta) to increase rigidity and favorable interactions of residues in highly flexible regions. MD results show that five regions have the highest flexibilities and were selected for design by Rosetta. Twenty-one designed mutants with the best ΔΔG at each position and ΔΔG < 0 REU were simulated at high temperature. Eight designed mutants with ΔRMSF of highly flexible regions lower than -10.0% were further simulated at the optimum temperature of the wild type. N88Q, N202V, G207D, Q209M, N211T and Y213K mutants were predicted to be more stable and could maintain their native structures better than the wild type due to increased hydrogen bond interactions of designed residues and their neighboring residues. These designed mutants are promising enzymes with high potential for stability improvement.

Molecular properties and ligand specificity of zebrafish intelectin-2.

Intelectins are immune lectins expressed in chordates, including several fish species, in which intelectins are known to be upregulated upon infection. However, the basic biochemical properties and bacteria binding specificities of several fish intelectins are not well studied. We focus our investigation on zebrafish intelectin-2 (DrIntL-2) that is predominantly expressed in the gastrointestinal tract. The disulfide-linked oligomeric state and the cysteine responsible for intermolecular disulfide bonds are identified. DrIntL-2 is a globular particle of around 30 nm. In addition to the typical exocyclic 1,2-diol ligands, DrIntL-2 binds ß-1,3-glucan and recognizes Salmonella typhimurium and Pseudomonas aeruginosa. This investigation not only shed light on the fish innate immunity that will be essential for the aquaculture industry, but will also provide a foundation for further application of DrIntL-2 in bacteria detection and identification.

Global diversity of the gene encoding the Pfs25 protein-a Plasmodium falciparum transmission-blocking vaccine candidate.

BACKGROUND: Vaccines against the sexual stages of the malarial parasite Plasmodium falciparum are indispensable for controlling malaria and abrogating the spread of drug-resistant parasites. Pfs25, a surface antigen of the sexual stage of P. falciparum, is a leading candidate for transmission-blocking vaccine development. While clinical trials have reported that Pfs25-based vaccines are safe and effective in inducing transmission-blocking antibodies, the extent of the genetic diversity of Pfs25 in malaria endemic populations has rarely been studied. Thus, this study aimed to investigate the global diversity of Pfs25 in P. falciparum populations. METHODS: A database of 307 Pfs25 sequences of P. falciparum was established. Population genetic analyses were performed to evaluate haplotype and nucleotide diversity, analyze haplotypic distribution patterns of Pfs25 in different geographical populations, and construct a haplotype network. Neutrality tests were conducted to determine evidence of natural selection. Homology models of the Pfs25 haplotypes were constructed, subjected to molecular dynamics (MD), and analyzed in terms of flexibility and percentages of secondary structures. RESULTS: The Pfs25 gene of P. falciparum was found to have 11 unique haplotypes. Of these, haplotype 1 (H1) and H2, the major haplotypes, represented 70% and 22% of the population, respectively, and were dominant in Asia, whereas only H1 was dominant in Africa, Central America, and South America. Other haplotypes were rare and region-specific, resulting in unique distribution patterns in different geographical populations. The diversity in Pfs25 originated from ten single-nucleotide polymorphism (SNP) loci located in the epidermal growth factor (EGF)-like domains and anchor domain. Of these, an SNP at position 392 (GGA/GCA), resulting in amino acid substitution 131 (Gly/Ala), defined the two major haplotypes. The MD results showed that the structures of H1 and H2 variants were relatively similar. Limited polymorphism in Pfs25 could likely be due to negative selection. CONCLUSIONS: The study successfully established a Pfs25 sequence database that can become an essential tool for monitoring vaccine efficacy, designing assays for detecting malaria carriers, and conducting epidemiological studies of P. falciparum. The discovery of the two major haplotypes, H1 and H2, and their conserved structures suggests that the current Pfs25-based vaccines could be used globally for malaria control.

Computational redesign of Fab CC12.3 with substantially better predicted binding affinity to SARS-CoV-2 than human ACE2 receptor.

SARS-CoV-2 is responsible for COVID-19 pandemic, causing large numbers of cases and deaths. It initiates entry into human cells by binding to the peptidase domain of angiotensin-converting enzyme 2 (ACE2) receptor via its receptor binding domain of S1 subunit of spike protein (SARS-CoV-2-RBD). Employing neutralizing antibodies to prevent binding between SARS-CoV-2-RBD and ACE2 is an effective COVID-19 therapeutic solution. Previous studies found that CC12.3 is a highly potent neutralizing antibody that was isolated from a SARS-CoV-2 infected patient, and its Fab fragment (Fab CC12.3) bound to SARS-CoV-2-RBD with comparable binding affinity to ACE2. To enhance its binding affinity, we employed computational protein design to redesign all CDRs of Fab CC12.3 and molecular dynamics (MD) to validate their predicted binding affinities by the MM-GBSA method. MD results show that the predicted binding affinities of the three best designed Fabs CC12.3 (CC12.3-D02, CC12.3-D05, and CC12.3-D08) are better than those of Fab CC12.3 and ACE2. Additionally, our results suggest that enhanced binding affinities of CC12.3-D02, CC12.3-D05, and CC12.3-D08 are caused by increased SARS-CoV-2-RBD binding interactions of CDRs L1 and L3. This study redesigned neutralizing antibodies with better predicted binding affinities to SARS-CoV-2-RBD than Fab CC12.3 and ACE2. They are promising candidates as neutralizing antibodies against SARS-CoV-2.

Computational design of SARS-CoV-2 peptide binders with better predicted binding affinities than human ACE2 receptor.

SARS-CoV-2 is coronavirus causing COVID-19 pandemic. To enter human cells, receptor binding domain of S1 subunit of SARS-CoV-2 (SARS-CoV-2-RBD) binds to peptidase domain (PD) of angiotensin-converting enzyme 2 (ACE2) receptor. Employing peptides to inhibit binding between SARS-CoV-2-RBD and ACE2-PD is a therapeutic solution for COVID-19. Previous experimental study found that 23-mer peptide (SBP1) bound to SARS-CoV-2-RBD with lower affinity than ACE2. To increase SBP1 affinity, our previous study used residues 21-45 of α1 helix of ACE2-PD (SPB25) to design peptides with predicted affinity better than SBP1 and SPB25 by increasing interactions of residues that do not form favorable interactions with SARS-CoV-2-RBD. To design SPB25 with better affinity than ACE2, we employed computational protein design to increase interactions of residues reported to form favorable interactions with SARS-CoV-2-RBD and combine newly designed mutations with the best single mutations from our previous study. Molecular dynamics show that predicted binding affinities of three peptides (SPB25Q22R, SPB25F8R/K11W/L25R and SPB25F8R/K11F/Q22R/L25R) are better than ACE2. Moreover, their predicted stabilities may be slightly higher than SBP1 as suggested by their helicities. This study developed an approach to design SARS-CoV-2 peptide binders with predicted binding affinities better than ACE2. These designed peptides are promising candidates as SARS-CoV-2 inhibitors.

Unravelling Regioselectivity of Leuconostoc citreum ABK-1 Alternansucrase by Acceptor Site Engineering.

Alternansucrase (ALT, EC 2.4.1.140) is a glucansucrase that can generate α-(1,3/1,6)-linked glucan from sucrose. Previously, the crystal structure of the first alternansucrase from Leuconostoc citreum NRRL B-1355 was successfully elucidated; it showed that alternansucrase might have two acceptor subsites (W675 and W543) responsible for the formation of alternating linked glucan. This work aimed to investigate the primary acceptor subsite (W675) by saturated mutagenesis using Leuconostoc citreum ABK-1 alternansucrase (LcALT). The substitution of other residues led to loss of overall activity, and formation of an alternan polymer with a nanoglucan was maintained when W675 was replaced with other aromatic residues. Conversely, substitution by nonaromatic residues led to the synthesis of oligosaccharides. Mutations at W675 could potentially cause LcALT to lose control of the acceptor molecule binding via maltose-acceptor reaction-as demonstrated by results from molecular dynamics simulations of the W675A variant. The formation of α-(1,2), α-(1,3), α-(1,4), and α-(1,6) linkages were detected from products of the W675A mutant. In contrast, the wild-type enzyme strictly synthesized α-(1,6) linkage on the maltose acceptor. This study examined the importance of W675 for transglycosylation, processivity, and regioselectivity of glucansucrases. Engineering glucansucrase active sites is one of the essential approaches to green tools for carbohydrate modification.

Biochemical and Structural Investigation of GnnA in the Lipopolysaccharide Biosynthesis Pathway of Acidithiobacillus ferrooxidans.

Lipopolysaccharide (LPS) is a crucial component in the outer membrane of Gram-negative bacteria that contributes to both pathogenicity as well as immunity against pathogenic bacteria. Typical LPS contains GlcN disaccharide as the core of lipid A. However, some bacteria such as Acidithiobacillus ferrooxidans and Leptospira interrogans contain GlcN3N in lipid A instead. This modification has been shown to dampen the host immune response and increase resistance to antimicrobial peptides. Therefore, investigation of the enzymes responsible for the biosynthesis of GlcN3N has promising applications in the development of vaccines, antibiotics, or usage of the enzymes in chemoenzymatic synthesis of modified LPS. Here, we describe biochemical and structural investigation of GnnA from A. ferrooxidans (AfGnnA) that is responsible for oxidation of UDP-GlcNAc, which subsequently undergoes transamination to produce UDP-GlcNAc3N as a precursor for LPS biosynthesis. AfGnnA is specific for NAD+ and UDP-GlcNAc. The crystal structures of AfGnnA in combination with molecular dynamics simulation and mutational analysis suggest the substrate recognition mode and the catalytic mechanism. K91 or H164 is a potential catalytic base in the oxidation reaction. The results will not only provide insights into the biosynthesis of unusual LPS but will also lay the foundation for development of more immunogenic vaccines, novel antibiotics, or utilization of GnnA in the synthesis of UDP-sugars or modified LPS.

Computational Design of 25-mer Peptide Binders of SARS-CoV-2.

SARS-CoV-2 is the novel coronavirus causing the COVID-19 pandemic. To enter human cells, the receptor-binding domain (RBD) of the S1 subunit of SARS-CoV-2 (SARS-CoV-2-RBD) initially binds to the peptidase domain of angiotensin-converting enzyme 2 receptor (ACE2-PD). Using peptides to inhibit SARS-CoV-2-RBD binding to ACE2 is a potential therapeutic solution for COVID-19. A previous study identified a 23-mer peptide (SBP1) that bound to SARS-CoV-2-RBD with comparable KD to ACE2. We employed computational protein design and molecular dynamics (MD) to design SARS-CoV-2-RBD 25-mer peptide binders (SPB25) with better predicted binding affinity than SBP1. Using residues 21-45 of the α1 helix of ACE2-PD as the template, our strategy is employing Rosetta to enhance SPB25 binding affinity to SARS-CoV-2-RBD and avoid disrupting existing favorable interactions by using residues that have not been reported to form favorable interactions with SARS-CoV-2-RBD as designed positions. Designed peptides with better predicted binding affinities, by Rosetta, than SPB25 were subjected to MD validation. The MD results show that five designed peptides (SPB25F8N, SPB25F8R, SPB25L25R, SPB25F8N/L25R, and SPB25F8R/L25R) have better predicted binding affinities, by the MM-GBSA method, than SPB25 and SBP1. This study developed an approach to design SARS-CoV-2-RBD peptide binders, and these peptides may be promising candidates as potential SARS-CoV-2 inhibitors.

Computational design of Bacillus licheniformis RN-01 levansucrase for control of the chain length of levan-type fructooligosaccharides.

Levansucrase (LS) from Gram-positive bacteria generally produces a large quantity of levan polymer, a polyfructose with glucose at the end (GFn) but a small quantity of levan-type fructooligosaccharides (LFOs). The properties of levan and LFOs depend on their chain lengths, thereby determining their potential applications in food and pharmaceutical industries such as prebiotics and anti-tumor agents. Therefore, an ability to redesign and engineer the active site of levansucrase for synthesis of products with desired degree of polymerization (DP) is very beneficial. We employed computational protein design, docking and molecular dynamics to redesign and engineer the active site of Bacillus licheniformis RN-01 levansucrase for production of LFOs with DP up to five (GF4), using two approaches: 1) blocking oligosaccharide binding track of GF3-LS complex with large aromatic residues and 2) eliminating hydrogen bond interactions between terminal glucose of GF4 and side chains of binding residues of GF4-LS complex. The designed enzymes and their product patterns from these two approaches were experimentally characterized. The experimental results show that the first approach was successful in creating N251W and N251W/K372Y mutants that synthesized LFOs with DP up to five. This work illustrates how computer-aided approaches can offer novel opportunities to engineer enzymes for desired products.

Modulation of fructooligosaccharide chain length and insight into the product binding motif of Lactobacillus reuteri 121 inulosucrase.

Inulosucrase (E.C. 2.4.1.9) is a bacterial fructosyltransferase that synthesizes inulin-type fructooligosaccharide, using sucrose as a substrate. We modulated the size of fructooligosaccharide synthesized by Lactobacillus reuteri 121 inulosucrase using rational designed mutagenesis. Nine residues: D478, D479, S482, R483, N543, W551, N555, N561 and D689, were changed based on the active site architecture and amino acids potentially interacting with saccharides. The selected residues were substituted with alanine to investigate the contribution of these residues to FOS chain length. Enzymatic activity assays demonstrated that the transglycosylation/hydrolysis ratios of D479A, R483A, N543A, W551A and N555A mutants were significantly different from that of the wild type. Almost all mutants, except D478A, synthesized oligosaccharides with different size distribution compared to that of wild type. Molecular docking further provides insights into the product binding motif of Lactobacillus reuteri 121 inulosucrase and strengthens an important role of amino acid residues at remote locations from the active site on the enzymatic activity and product specificity.

Molecular dynamics provides insight into how N251A and N251Y mutations in the active site of Bacillus licheniformis RN-01 levansucrase disrupt production of long-chain levan.

Produced by levansucrase, levan and levan oligosaccharides (GFn) have potential applications in food and pharmaceutical industries such as prebiotics, anti-tumor and anti-inflammatory agents. Previous study reported that Bacillus licheniformis RN-01 levansucrase could produce levan oligosaccharides and long-chain levan. However, its N251A and N251Y mutants could effectively produce short-chain oligosaccharides upto GF3, but they could not produce long-chain levan. We hypothesized that these mutations probably reduced GF3 binding affinity in levansucrase active site that contains fructosyl-Asp93 intermediate and caused GF3 to be in an unfavorable orientation for transfructosylation; therefore, levansucrase could not effectively extend GF3 by one fructosyl residue to produce GF4 and subsequently long-chain levan. However, these mutations probably did not significantly reduce binding affinity or drastically change orientation of GF2; therefore, levansucrase could still extend GF2 to produce GF3. Using this hypothesis, we employed molecular dynamics to investigate effects of these mutations on GF2/GF3 binding in levansucrase active site. Our results reasonably support this hypothesis as N251A and N251Y mutations did not significantly reduce GF2 binding affinity, as calculated by MM-GBSA technique and hydrogen bond occupations, or drastically change orientation of GF2 in levansucrase active site, as measured by distance between atoms necessary for transfructosylation. However, these mutations drastically decreased GF3 binding affinity and caused GF3 to be in an unfavorable orientation for transfructosylation. Furthermore, the free energy decomposition and hydrogen bond occupation results suggest the importance of Arg255 in GF2/GF3 binding in levansucrase active site. This study provides important and novel insight into the effects of N251A and N251Y mutations on GF2/GF3 binding in levansucrase active site and how they may disrupt production of long-chain levan. This knowledge could be beneficial in designing levansucrase to efficiently produce levan oligosaccharides with desired length.