Yaming Shan

Insight into the interactive residues between two domains of human somatic Angiotensin-converting enzyme and Angiotensin II by MM-PBSA calculation and steered molecular dynamics simulation

Angiotensin-converting enzyme (ACE), a membrane-bound zinc metallopeptidase, catalyzes the formation of Angiotensin-II (AngII) and the deactivation of bradykinin in the renin–angiotensin-aldosterone and kallikrein–kinin systems. As a hydrolysis product of ACE, AngII is regarded as an inhibitor and displays stronger competitive inhibition in the C-domain than the N-domain of ACE. However, the AngII binding differences between the two domains and the mechanisms behind AngII dissociation from the C-domain are rarely explored. In this work, molecular docking, Molecular Mechanics/Poisson–Boltzmann Surface Area calculation, and steered molecular dynamics (SMD) are applied to explore the structures and interactions in the binding or unbinding of AngII with the two domains of human somatic ACE. Calculated free energy values suggest that the C-domain–AngII complex is more stable than the N-domain–AngII complex, consistent with available experimental data. SMD simulation results imply that electrostatic interaction is dominant in the dissociation of AngII from the C-domain. Moreover, Gln106, Asp121, Glu123, and Tyr213 may be the key residues in the unbinding pathway of AngII. The simulation results in our work provide insights into the interactions between the two domains of ACE and its natural peptide inhibitor AngII at a molecular level. Moreover, the results provide theoretical clues for the design of new inhibitors.

Multiple antigen peptide mimetics containing gp41 membrane-proximal external region elicit broad neutralizing antibodies against human immunodeficiency virus type 1 in guinea pigs

Eliciting a broadly neutralizing antibody response against the HIV‐1 membrane‐proximal external region (MPER) mimicking the activity of 4E10 and 2F5 monoclonal antibodies remains a major challenge. In this study, two novel tetra‐branched peptide immunogens, 4E10‐ and 2F5‐MAP4, were designed and synthesized using a MAP system. Guinea pigs were immunized with either of these two synthetic immunogens emulsified in an oil‐phase adjuvant at 3‐week intervals. After four immunizations, epitope‐specific antibody responses were induced successfully, and moderate neutralizing activities against tier 1 (clades B, BC, AE) and tier 2 (clade C) HIV‐1 pseudoviruses were detectable in unfractionated sera and purified IgGs. The synthetic gp41 membrane‐proximal external region peptide mimetics, 4E10‐ and 2F5‐MAP4, assisted by an appropriate adjuvant, are promising prophylactic vaccine candidates potentially capable of eliciting broadly neutralizing antibody responses against HIV‐1. Copyright

Structural Basis of Fullerene Derivatives as Novel Potent Inhibitors of Protein Tyrosine Phosphatase 1B: Insight into the Inhibitory Mechanism through Molecular Modeling Studies

Protein tyrosine phosphatase 1B (PTP1B) has become an outstanding target for the treatment of diabetes and obesity. Recent research has demonstrated that some fullerene derivatives serve as a new nanoscale-class of potent inhibitors of PTP1B, but the specific mechanism remains unclear. Several molecular modeling methods (molecular docking, molecular dynamics simulations, and molecular mechanics/generalized Born surface area calculations) were integrated to provide insight into the binding mode and inhibitory mechanism of the new class of fullerene inhibitors. The results reveal that PTP1B with an open WPD loop is more susceptible to the combination with the fullerene inhibitor because of their comparable shapes and sizes. When the WPD loop fluctuates to the open conformation, the inhibitor falls into the active pocket and induces conformational rotation of the WPD loop. This rotation is closely related to the reduction of the catalytic activity of PTP1B. In addition, it is suggested that compound 1, like compound 2, is a competitive inhibitor since it blocks the active site to prevent the binding of the substrate. The high binding affinity of fullerene-based compounds and the transition of the WPD loop, caused by the specific structural property of the hydrophobic fullerene core and the appended polar groups, make these fullerene derivatives efficient competitive inhibitors. The theoretical results provide useful clues for further investigation of the noval inhibitors of PTP1B at the nanoscale.

A novel modified peptide derived from membrane-proximal external region of human immunodeficiency virus type 1 envelope significantly enhances retrovirus infection

Efficient gene transfer is a critical goal in retroviral transduction. Several peptides capable of forming amyloid fibrils, such as the 39‐residue semen‐derived infection‐enhancing peptide (SEVI), have demonstrated the ability to boost retroviral gene delivery. Here, a 13‐residue peptide P13 (Ac‐671NWFDITNWLWYIK683) derived from the membrane‐proximal external region of the human immunodeficiency virus type 1 (HIV‐1) gp41 transmembrane protein, together with its 16‐residue peptide derivative (P16) were found to enhance HIV‐1 infection significantly. Both peptides, P13 and P16, could form amyloid fibril structures to potently enhance HIV‐1 infectivity. Further investigations showed that both aromatic Trp residues and cationic Lys residues contributed to the enhancement of HIV‐1 infection by these two active peptides. P16 could more effectively augment HIV‐1 YU‐2 infection than SEVI, implying its potential applications as a tool in the lab to improve gene transfer rates. Copyright

Structural and molecular basis of cellulase Cel48F by computational modeling: Insight into catalytic and product release mechanism.

As a processive cellulase, Cel48F from Clostridium cellulolyticum plays a crucial role in cellulose fiber degradation. It has been confirmed in experiment that residue Glu44 will greatly affect the catalytic activity but the mechanism is still unknown. In this study, conventional molecular dynamics, steered molecular dynamics and free energy calculation were integrated to simulate the hydrolysis and product release process to gain insights into the factors that influence catalytic activity. Analysis of simulation results indicated that Glu44 could maintain the proper conformation of its substrate to ensure successful cleavage reaction or serve as a base required in the inverting mechanism in hydrolysis. After hydrolysis is completed, residues Glu44, Asp494, Trp611 and Glu55 participate in hydrogen bond rearrangement during product releasing process. This rearrangement can reduce the sliding barrier and stimulate the product to move toward the exit in the initial release stage. Dependent on the rearrangement, the product moves toward the exit and is exposed to an increasing amount of solvent molecules, which makes solvent effect more and more notable. With the assistance of solvent interaction, product can get rid of the enzyme more easily. However, the subsequent release process remains uncertain because of the disordered motion of solvent molecules. This work provides theoretical data as a basis of cellulase modification or mutation.

Conserved stem fragment from H3 influenza hemagglutinin elicits cross-clade neutralizing antibodies through stalk-targeted blocking of conformational change during membrane fusion

Currently available influenza vaccines typically fail to elicit/boost broadly neutralizing antibodies due to the mutability of virus sequences and conformational changes during protective immunity, thereby limiting their efficacy. This problem needs to be addressed by further understanding the mechanisms of neutralization and finding the desired neutralizing site during membrane fusion. This study specifically focused on viruses of the H3N2 subtype, which have persisted as a principal source of influenza-related morbidity and mortality in humans since the 1968 influenza pandemic. Through sequence alignment and epitope prediction, a series of highly conserved stem fragments (spanning 47 years) were found and coupled to the Keyhole Limpet Hemocyanin (KLH) protein. By application of a combinatorial display library and crystal structure modeling, a stem fragment immunogen, located at the turning point of the HA neck undergoing conformational change during membrane fusion with both B- and T-cell epitopes, was identified. After synthesis of the optimal stem fragment using a multiple antigen peptide (MAP) system, strong humoral immune responses and cross-clade neutralizing activities against strains from the H3 subtype of group 2 influenza viruses after animal immunizations were observed. By detection of nuclear protein immunofluorescence with acid bypass treatment, antisera raised against MAP4 immunogens of the stem fragment showed the potential to inhibit the conformational change of HA in stem-targeted virus neutralization. The identification of this conserved stem fragment provides great potential for exploitation of this site of vulnerability in therapeutic and vaccine design.

Elicitation of HIV-1 neutralizing antibodies by presentation of 4E10 and 10E8 epitopes on Norovirus P particles.

Eliciting efficient broadly neutralizing antibodies (BnAbs) is a major goal in vaccine development against human immunodeficiency virus type 1 (HIV-1). Conserved epitopes in the membrane-proximal external region (MPER) of HIV-1 are a significant target. In this study, Norovirus P particles (NoV PPs) were used as carriers to display conformational 4E10 and 10E8 epitopes in different patterns with an appropriate linker. Immune responses to the recombinant NoV PPs were characterized in guinea pigs and Balb/c mice and could induce high levels of MPER-binding antibodies. Modest neutralizing activities could be detected in sera of guinea pigs but not of Balb/c mice. The 4E10 or 10E8 epitopes dispersed on three loops on the outermost surface of NoV PPs (4E10-loop123 PP or 10E8-loop123 PP) elicited higher neutralizing activities than the equivalent number of epitopes presented on loop 2 only (4E10-3loop2 PP or 10E8-3loop2 PP). The epitopes on different loops of the PP were well-exposed and likely formed an appropriate conformation to induce neutralizing antibodies. Although sera of immunized guinea pigs could neutralize several HIV envelope-pseudoviruses, a vaccine candidate for efficiently inducing HIV-1 BnAbs remains to be developed.

Exploration of binding and inhibition mechanism of a small molecule inhibitor of influenza virus H1N1 hemagglutinin by molecular dynamics simulation

Influenza viruses are a major public health threat worldwide. The influenza hemagglutinin (HA) plays an essential role in the virus life cycle. Due to the high conservation of the HA stem region, it has become an especially attractive target for inhibitors for therapeutics. In this study, molecular simulation was applied to study the mechanism of a small molecule inhibitor (MBX2329) of influenza HA. Behaviors of the small molecule under neutral and acidic conditions were investigated, and an interesting dynamic binding mechanism was found. The results suggested that the binding of the inhibitor with HA under neutral conditions facilitates only its intake, while it interacts with HA under acidic conditions using a different mechanism at a new binding site. After a series of experiments, we believe that binding of the inhibitor can prevent the release of HA1 from HA2, further maintaining the rigidity of the HA2 loop and stabilizing the distance between the long helix and short helices. The investigated residues in the new binding site show high conservation, implying that the new binding pocket has the potential to be an effective drug target. The results of this study will provide a theoretical basis for the mechanism of new influenza virus inhibitors.

A simple colorimetric and fluorescent probe with high selectivity towards cysteine over homocysteine and glutathione

A novel fluorescent probe (AQDA) based on quinizarin is designed and synthesized. Owing to a nucleophilic addition and a specific intramolecular cyclization reaction, the probe displays high selectivity towards cysteine (Cys) relative to other natural amino acids. The maximum fluorescent intensity is 30-fold that of the initial value in the presence of 5.0 equiv. Cys, and its detection limit is 0.158 μM. The recognition mechanism is further confirmed through mass spectroscopy and proton nuclear magnetic resonance titration. Simultaneously, the fluorescence enhancement mechanism is characterized by theoretical calculations, and experimental data are consistent with the theoretical results. Finally, the cellular imaging experiment verifies that AQDA possesses the capacity to detect endogenous Cys in living cells.

Potential of a novel peptide P16-D from the membrane-proximal external region of human immunodeficiency virus type 1 to enhance retrovirus infection

The peptide P13 (Ac-671NWFDITNWLWYIK683-NH2), derived from the membrane-proximal external region (MPER) of the human immunodeficiency virus type 1 (HIV-1) transmembrane protein and its derivative P16, have been shown to significantly boost HIV-1 infectivity by forming amyloid fibrils. Here, a new modified nanofibril peptide P16-D derived from P16 was demonstrated to have an enhanced ability to promote retroviral gene transfer. Moreover, the “networks” formed by P16-D nanofibrils could effectively capture and concentrate enveloped virus by low-speed centrifugation. In addition, the captured influenza virus H1N1 could elicit a stronger immune response in mice at a lower dose than that in the absence of the nanofibrils. The results implied a potential for P16-D to improve gene transfer rates and vaccine applications.