Characterizing the Diversity of the CDR-H3 Loop Conformational Ensembles in Relationship to Antibody Binding Properties

We present an approach to assess antibody CDR-H3 loops according to their dynamic properties using molecular dynamics simulations. We selected six antibodies in three pairs differing substantially in their individual promiscuity respectively specificity. For two pairs of antibodies crystal structures are available in different states of maturation and used as starting structures for the analyses. For a third pair we chose two antibody CDR sequences obtained from a synthetic library and predicted the respective structures. For all three pairs of antibodies we performed metadynamics simulations to overcome the limitations in conformational sampling imposed by high energy barriers. Additionally, we used classic molecular dynamics simulations to describe nano- to microsecond flexibility and to estimate up to millisecond kinetics of captured conformational transitions. The methodology represents the antibodies as conformational ensembles and allows comprehensive analysis of structural diversity, thermodynamics of conformations and kinetics of structural transitions. Referring to the concept of conformational selection we investigated the link between promiscuity and flexibility of the antibodies' binding interfaces. The obtained detailed characterization of the binding interface clearly indicates a link between structural flexibility and binding promiscuity for this set of antibodies

Supplement: "Localization and broadband follow-up of the gravitational-wave transient GW150914" (2016, ApJL, 826, L13)

This Supplement provides supporting material for Abbott et al. (2016a). We briefly summarize past electromagnetic (EM) follow-up efforts as well as the organization and policy of the current EM follow-up program. We compare the four probability sky maps produced for the gravitational-wave transient GW150914, and provide additional details of the EM follow-up observations that were performed in the different bands

Pore mutation N617D in the skeletal muscle DHPR blocks Ca2+ influx due to atypical high-affinity Ca2+ binding

Skeletal muscle excitation-contraction (EC) coupling roots in Ca2+-influx-independent inter-channel signaling between the sarcolemmal dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR1) in the sarcoplasmic reticulum. Although DHPR Ca2+ influx is irrelevant for EC coupling, its putative role in other muscle-physiological and developmental pathways was recently examined using two distinct genetically engineered mouse models carrying Ca2+ non-conducting DHPRs: DHPR(N617D) (Dayal et al., 2017) and DHPR(E1014K) (Lee et al., 2015). Surprisingly, despite complete block of DHPR Ca2+-conductance, histological, biochemical, and physiological results obtained from these two models were contradictory. Here, we characterize the permeability and selectivity properties and henceforth the mechanism of Ca2+ non-conductance of DHPR(N617). Our results reveal that only mutant DHPR(N617D) with atypical high-affinity Ca2+ pore-binding is tight for physiologically relevant monovalent cations like Na+ and K+. Consequently, we propose a molecular model of cooperativity between two ion selectivity rings formed by negatively charged residues in the DHPR pore region

Water model determines thermosensitive and physicochemical properties of poly(N-isopropylacrylamide) in molecular simulations

Poly (N-isopropylacrylamide) (PNIPAM) is a famous representative of thermosensitive polymers. Thermosensitive polymers undergo a phase transition with lower critical solution temperature. Commonly, their phase behavior is linked to a conformational collapse above a certain temperature. This thermosensitive conformational transition is called Coil-Globule transition. In contrast, most other polymers usually show inverse temperature behavior, i.e., an upper critical solution temperature, corresponding to a Globule-Coil transition. Besides their numerous possible applications, thermosensitive polymers are of interest for fundamental research, because of similarities to macromolecular conformational transitions, e.g., protein folding. The counter-intuitive behavior of thermosensitive polymers is commonly associated with solvation effects. Thus, an accurate description of the solvent is crucial for the investigation of thermosensitive polymers in molecular simulations. Here, we investigate the influence of the in silico water model on the thermosensitive Coil-Globule transition in molecular dynamics simulations. To this end, we performed extensive atomistic simulations of the syndiotactic PNIPAM 20-mer at multiple temperatures with eight different water models–four of which are 3-point water models (TIP3P-type) and four are 4-point water models (TIP4P-type). We found that the thermosensitive Coil-Globule transition is strongly influenced by the water model in the simulations. Depending on the water model, the conformational ensemble of the polymer is shifted significantly, which leads to dramatically different results: The estimated transition temperature may span between 255 and 350 K. Consequently, depending on the description of the solvent, the physicochemical and mechanical properties of these polymers, e.g., the polymer-solvent affinity and persistence length, vary. These divergent results originate from the strength of interactions between polymer and solvent, but also on the bulk state of the solvent. Both these quantities vary between water models. We found that the Lennard-Jones interaction parameter ϵ of the water model correlates with the transition temperature of the polymer. Indeed, the quadrupole moment of the water model shows an even higher correlation with this quantity. Our results suggest a connection between the phase diagram of the solvent and the thermosensitive transition of the polymer.ISSN:2296-801

Nanobody Paratope Ensembles in Solution Characterized by MD Simulations and NMR

Variable domains of camelid antibodies (so-called nanobodies or VHH) are the smallest antibody fragments that retain complete functionality and therapeutic potential. Understanding of the nanobody-binding interface has become a pre-requisite for rational antibody design and engineering. The nanobody-binding interface consists of up to three hypervariable loops, known as the CDR loops. Here, we structurally and dynamically characterize the conformational diversity of an anti-GFP-binding nanobody by using molecular dynamics simulations in combination with experimentally derived data from nuclear magnetic resonance (NMR) spectroscopy. The NMR data contain both structural and dynamic information resolved at various timescales, which allows an assessment of the quality of protein MD simulations. Thus, in this study, we compared the ensembles for the anti-GFP-binding nanobody obtained from MD simulations with results from NMR. We find excellent agreement of the NOE-derived distance maps obtained from NMR and MD simulations and observe similar conformational spaces for the simulations with and without NOE time-averaged restraints. We also compare the measured and calculated order parameters and find generally good agreement for the motions observed in the ps–ns timescale, in particular for the CDR3 loop. Understanding of the CDR3 loop dynamics is especially critical for nanobodies, as this loop is typically critical for antigen recognition

Addressing Challenges of Macrocyclic Conformational Sampling in Polar and Apolar Solvents: Lessons for Chameleonicity

We evaluated a workflow to reliably sample the conformational space of a set of 47 peptidic macrocycles. Starting from SMILES strings, we use accelerated molecular dynamics simulations to overcome high energy barriers, in particular, the cis-trans isomerization of peptide bonds. We find that our approach performs very well in polar solvents like water and dimethyl sulfoxide. Interestingly, the protonation state of a secondary amine in the ring only slightly influences the conformational ensembles of our test systems. For several of the macrocycles, determining the conformational distribution in chloroform turns out to be considerably more challenging. Especially, the choice of partial charges crucially influences the ensembles in chloroform. We address these challenges by modifying initial structures and the choice of partial charges. Our results suggest that special care has to be taken to understand the configurational distribution in apolar solvents, which is a key step toward a reliable prediction of membrane permeation of macrocycles and their chameleonic properties.ISSN:1549-9596ISSN:0095-2338ISSN:1520-514

The Structural Flexibility of PR-10 Food Allergens

PR-10 proteins constitute a major cause of food allergic reactions. Birch-pollen-related food allergies are triggered by the immunologic cross-reactivity of IgE antibodies with structurally homologous PR-10 proteins that are present in birch pollen and various food sources. While the three-dimensional structures of PR-10 food allergens have been characterized in detail, only a few experimental studies have addressed the structural flexibility of these proteins. In this study, we analyze the millisecond-timescale structural flexibility of thirteen PR-10 proteins from prevalent plant food sources by NMR relaxation-dispersion spectroscopy, in a comparative manner. We show that all the allergens in this study have inherently flexible protein backbones in solution, yet the extent of the structural flexibility appears to be strikingly protein-specific (but not food-source-specific). Above-average flexibility is present in the two short helices, α1 and α2, which form a V-shaped support for the long C-terminal helix α3, and shape the internal ligand-binding cavity, which is characteristic for PR-10 proteins. An in-depth analysis of the NMR relaxation-dispersion data for the PR-10 allergen from peanut reveals the presence of at least two subglobal conformational transitions on the millisecond timescale, which may be related to the release of bound low-molecular-weight ligands from the internal cavity

Characterization of two pathological gating-charge substitutions in Cav1.4 L-type calcium channels

Cav1.4 L-type calcium channels are predominantly expressed at the photoreceptor terminals and in bipolar cells, mediating neurotransmitter release. Mutations in its gene, CACNA1F, can cause congenital stationary night-blindness type 2 (CSNB2). Due to phenotypic variability in CSNB2, characterization of pathological variants is necessary to better determine pathological mechanism at the site of action. A set of known mutations affects conserved gating charges in the S4 voltage sensor, two of which have been found in male CSNB2 patients. Here, we describe two disease-causing Cav1.4 mutations with gating charge neutralization, exchanging an arginine 964 with glycine (RG) or arginine 1288 with leucine (RL). In both, charge neutralization was associated with a reduction channel expression also reflected in smaller ON gating currents. In RL channels, the strong decrease in whole-cell current densities might additionally be explained by a reduction of single-channel currents. We further identified alterations in their biophysical properties, such as a hyperpolarizing shift of the activation threshold and an increase in slope factor of activation and inactivation. Molecular dynamic simulations in RL substituted channels indicated water wires in both, resting and active, channel states, suggesting the development of omega (ω)currents as a new pathological mechanism in CSNB2. This sum of the respective channel property alterations might add to the differential symptoms in patients beside other factors, such as genomic and environmental deviations.</p

High Intrinsic Oncogenic Potential in the Myc-Box-Deficient <i>Hydra</i> Myc3 Protein

The proto-oncogene myc has been intensively studied primarily in vertebrate cell culture systems. Myc transcription factors control fundamental cellular processes such as cell proliferation, cell cycle control and stem cell maintenance. Myc interacts with the Max protein and Myc/Max heterodimers regulate thousands of target genes. The genome of the freshwater polyp Hydra encodes four myc genes (myc1-4). Previous structural and biochemical characterization showed that the Hydra Myc1 and Myc2 proteins share high similarities with vertebrate c-Myc, and their expression patterns suggested a function in adult stem cell maintenance. In contrast, an additional Hydra Myc protein termed Myc3 is highly divergent, lacking the common N-terminal domain and all conserved Myc-boxes. Single cell transcriptome analysis revealed that the myc3 gene is expressed in a distinct population of interstitial precursor cells committed to nerve- and gland-cell differentiation, where the Myc3 protein may counteract the stemness actions of Myc1 and Myc2 and thereby allow the implementation of a differentiation program. In vitro DNA binding studies showed that Myc3 dimerizes with Hydra Max, and this dimer efficiently binds to DNA containing the canonical Myc consensus motif (E-box). In vivo cell transformation assays in avian fibroblast cultures further revealed an unexpected high potential for oncogenic transformation in the conserved Myc3 C-terminus, as compared to Hydra Myc2 or Myc1. Structure modeling of the Myc3 protein predicted conserved amino acid residues in its bHLH-LZ domain engaged in Myc3/Max dimerization. Mutating these amino acid residues in the human c-Myc (MYC) sequence resulted in a significant decrease in its cell transformation potential. We discuss our findings in the context of oncogenic transformation and cell differentiation, both relevant for human cancer, where Myc represents a major driver