In silico design of high-affinity antigenic peptides for HLA-B44.

Cancer cell-killing by CD8+ T cells demands effective tumor antigen presentation by human leukocyte antigen class I (HLA-I) molecules. Screening and designing highly immunogenic neoantigens require quantitative computations to reliably predict HLA-peptide binding affinities. Here, with all-atom molecular dynamics (MD) simulations and free energy perturbation (FEP) methods, we design a collection of antigenic peptide candidates through in silico mutagenesis studies on immunogenic neoantigens, yielding enhanced binding affinities to HLA-B*44:02. In-depth structural dissection shows that introducing positively charged residues such as arginine to position 6 or lysine to position 7 of the candidates triggers conformational shifts in both peptides and the antigen-binding groove of the HLA, following the "induced-fit" mechanism. Enhancement in binding affinities compared to the wild-type was found in three out of five mutated candidates. The HLA pocket, capable of accommodating positively charged residues in positions from 5 to 7, is designated as the "dynamic pocket". Taken together, we showcase an effective structure-based binding affinity optimization framework for antigenic peptides of HLA-B*44:02 and underscore the importance of dynamic nature of the antigen-binding groove in concert with the anchoring motifs. This work provides structural insights for rational design of favorable HLA-peptide bindings and future developments in neoantigen-based therapeutics.

Ribonucleic Acid Folding Prediction Based on Iterative Multiscale Simulation.

RNA folding prediction is a challenge. Currently, many RNA folding models are coarse-grained (CG) with the potential derived from the known RNA structures. However, this potential is not suitable for modified and entirely new RNA. It is also not suitable for the folding simulation of RNA in the real cellular environment, including many kinds of molecular interactions. In contrast, our proposed model has the potential to address these issues, which is a multiscale simulation scheme based on all-atom (AA) force fields. We fit the CG force field using the trajectories generated by the AA force field and then iteratively perform molecular dynamics (MD) simulations of the two scales. The all-atom molecular dynamics (AAMD) simulation is mainly responsible for the correction of RNA structure, and the CGMD simulation is mainly responsible for efficient conformational sampling. On the basis of this scheme, we can successfully fold three RNAs belonging to a hairpin, a pseudoknot, and a four-way junction.

Adaptively Iterative Multiscale Switching Simulation Strategy and Applications to Protein Folding and Structure Prediction.

Structure prediction is an important means to quickly understand new protein functions. However, the prediction of effects of proteins that have no detectable templates is still to be improved. Molecular dynamics simulation is supposed to be the primary research tool for structure predictions, but it still has limitations of huge computational cost in all-atom (AA) models and rough accuracy in coarse-grained (CG) models. We propose a universal multiscale simulation strategy named AIMS in which simulations can iteratively switch among multiple resolutions in order to adaptively trade off AA accuracy and CG high-efficiency. AIMS follows the idea of CG-guided enhanced sampling so that final results always keep AA accuracy. We successfully achieve four ab initio and four data-assisted protein structure predictions using AIMS. The prediction result is an ensemble rather than a structure and provides special insights on folding metastable states. AIMS is estimated to achieve a computational speed about 40 times faster than that of conventional AA simulations.

A novel partially open state of SHP2 points to a "multiple gear" regulation mechanism.

The protein tyrosine phosphatase SHP2 mediates multiple signal transductions in various cellular pathways, controlled by a variety of upstream inputs. SHP2 dysregulation is causative of different types of cancers and developmental disorders, making it a promising drug target. However, how SHP2 is modulated by its different regulators remains largely unknown. Here, we use single-molecule fluorescence resonance energy transfer and molecular dynamics simulations to investigate this question. We identify a partially open, semiactive conformation of SHP2 that is intermediate between the known open and closed states. We further demonstrate a "multiple gear" regulatory mechanism, in which different activators (e.g., insulin receptor substrate-1 and CagA), oncogenic mutations (e.g., E76A), and allosteric inhibitors (e.g., SHP099) can shift the equilibrium of the three conformational states and regulate SHP2 activity to different levels. Our work reveals the essential role of the intermediate state in fine-tuning the activity of SHP2, which may provide new opportunities for drug development for relevant cancers.

Structural insights into telomere protection and homeostasis regulation by yeast CST complex.

Budding yeast Cdc13-Stn1-Ten1 (CST) complex plays an essential role in telomere protection and maintenance. Despite extensive studies, only structural information of individual domains of CST is available; the architecture of CST still remains unclear. Here, we report crystal structures of Kluyveromyces lactis Cdc13-telomeric-DNA, Cdc13-Stn1 and Stn1-Ten1 complexes and propose an integrated model depicting how CST assembles and plays its roles at telomeres. Surprisingly, two oligonucleotide/oligosaccharide-binding (OB) folds of Cdc13 (OB2 and OB4), previously believed to mediate Cdc13 homodimerization, actually form a stable intramolecular interaction. This OB2-OB4 module of Cdc13 is required for the Cdc13-Stn1 interaction that assembles CST into an architecture with a central ring-like core and multiple peripheral modules in a 2:2:2 stoichiometry. Functional analyses indicate that this unique CST architecture is essential for both telomere capping and homeostasis regulation. Overall, our results provide fundamentally valuable structural information regarding the CST complex and its roles in telomere biology.

Arbitrary Resolution with Two Bead Types Coarse-Grained Strategy and Applications to Protein Recognition.

Molecular recognition is a fundamental step in essentially any biological process. However, the kinetic processes during association and dissociation are difficult to be efficiently sampled by direct all-atom molecular dynamics simulations because of the large spatial and temporal scales. Here we propose an arbitrary resolution with two bead types (ART) coarse-grained (CG) strategy that is adept in molecular recognition. ART is a universal user-customized CG strategy that can generate a system-specific CG force field anytime and be applied to any system with an arbitrary CG resolution according to research requirements. ART CG simulations can be very efficiently performed with implicit solvation in prevalent simulation packages and provide interfaces for any enhanced sampling method. We used three applications, HLA-HIV epitope recognition, barnase-barstar association, and trimeric TRAF2 self-assembly, to validate the feasibility of the ART CG strategy, its advantages in protein recognition, and its high performance in simulations. Regular CG simulations can successfully achieve valid protein recognitions without any prior bound structure.

Different regions of synaptic vesicle membrane regulate VAMP2 conformation for the SNARE assembly.

Vesicle associated membrane protein 2 (VAMP2/synaptobrevin2), a core SNARE protein residing on synaptic vesicles (SVs), forms helix bundles with syntaxin-1 and SNAP25 for the SNARE assembly. Prior to the SNARE assembly, the structure of VAMP2 is unclear. Here, by using in-cell NMR spectroscopy, we describe the dynamic membrane association of VAMP2 SNARE motif in mammalian cells, and the structural change of VAMP2 upon the change of intracellular lipid environment. We analyze the lipid compositions of the SV membrane by mass-spectrometry-based lipidomic profiling, and further reveal that VAMP2 forms distinctive conformations in different membrane regions. In contrast to the non-raft region, the membrane region of cholesterol-rich lipid raft markedly weakens the membrane association of VAMP2 SNARE motif, which releases the SNARE motif and facilitates the SNARE assembly. Our work reveals the regulation of different membrane regions on VAMP2 structure and sheds light on the spatial regulation of SNARE assembly.

Structural and functional insights into the tetrameric photosystem I from heterocyst-forming cyanobacteria.

Two large protein-cofactor complexes, photosystem I and photosystem II, are the central components of photosynthesis in the thylakoid membranes. Here, we report the 2.37-Å structure of a tetrameric photosystem I complex from a heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Four photosystem I monomers, organized in a dimer of dimer, form two distinct interfaces that are largely mediated by specifically orientated polar lipids, such as sulfoquinovosyl diacylglycerol. The structure depicts a more closely connected network of chlorophylls across monomer interfaces than those seen in trimeric PSI from thermophilic cyanobacteria, possibly allowing a more efficient energy transfer between monomers. Our physiological data also revealed a functional link of photosystem I oligomerization to cyclic electron flow and thylakoid membrane organization.

Interwoven Molecular Chains Obtained by Ionic Self-Assembly of Two Iron(III) Porphyrins with Opposite and Mismatched Charges.

We report ionic self-assembly of positively charged FeIII meso-tetra(N-methyl-4-pyridyl) porphyrin (FeIIINMePyP) with negatively charged FeIII meso-tetra(4-sulfonatophenyl) porphyrin (FeIIITPPS4), leading to the formation of flower-like nanostructures composed of unprecedented three-dimensional (3D) entangled chains of porphyrin dimers. Molecular dynamics (MD) simulations show that the 3D entanglement of porphyrin chains closely correlates to mismatched charges present in porphyrin dimers like [FeIII(H2O)2NMePyP]5+/[FeIII(H2O)2TPPS4]3- that requires extra interactions or entanglement with neighboring ones to achieve electric neutrality. Interestingly, the interwoven chains bring in excellent thermal stability as evidenced by well maintenance of the flower-like morphology after pyrolysis at 775 °C in argon, which is in good agreement of high-temperature MD simulations. Meanwhile, heat treatment of the flower-like porphyrin nanostructure leads to the formation of a non-noble metal electrocatalyst (NNME) with largely inherited morphology. This exemplifies a new approach by combining ionic self-assembly with subsequent pyrolysis for the synthesis of NNMEs with desired control over the morphology of template-free NNMEs that has rarely been achieved prior to this study. Furthermore, our electrocatalyst exhibits excellent activity and durability toward oxygen reduction reaction as well as much better methanol tolerance compared with commercial Pt/C in alkaline solutions.

Integrin α4ß7 switches its ligand specificity via distinct conformer-specific activation.

Chemokine (C-C motif) ligand 25 (CCL25) and C-X-C motif chemokine 10 (CXCL10) induce the ligand-specific activation of integrin α4ß7 to mediate the selective adhesion of lymphocytes to mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1) or vascular cell adhesion molecule-1 (VCAM-1). However, the mechanism underlying the selective binding of different ligands by α4ß7 remains obscure. In this study, we demonstrate that CCL25 and CXCL10 induce distinct active conformers of α4ß7 with a high affinity for either MAdCAM-1 or VCAM-1. Single-cell force measurements show that CCL25 increases the affinity of α4ß7 for MAdCAM-1 but decreases its affinity for VCAM-1, whereas CXCL10 has the opposite effect. Structurally, CCL25 induces a more extended active conformation of α4ß7 compared with CXCL10-activated integrin. These two distinct intermediate open α4ß7 conformers selectively bind to MAdCAM-1 or VCAM-1 by distinguishing their immunoglobulin domain 2. Notably, Mn2+ fully opens α4ß7 with a high affinity for both ligands. Thus, integrin α4ß7 adopts different active conformations to switch its ligand-binding specificity.