Polyreactive Broadly Neutralizing B cells Are Selected to Provide Defense against Pandemic Threat Influenza Viruses.

Polyreactivity is the ability of a single antibody to bind to multiple molecularly distinct antigens and is a common feature of antibodies induced upon pathogen exposure. However, little is known about the role of polyreactivity during anti-influenza virus antibody responses. By analyzing more than 500 monoclonal antibodies (mAbs) derived from B cells induced by numerous influenza virus vaccines and infections, we found mAbs targeting conserved neutralizing influenza virus hemagglutinin epitopes were polyreactive. Polyreactive mAbs were preferentially induced by novel viral exposures due to their broad viral binding breadth. Polyreactivity augmented mAb viral binding strength by increasing antibody flexibility, allowing for adaption to imperfectly conserved epitopes. Lastly, we found affinity-matured polyreactive B cells were typically derived from germline polyreactive B cells that were preferentially selected to participate in B cell responses over time. Together, our data reveal that polyreactivity is a beneficial feature of antibodies targeting conserved epitopes.

Broadly neutralizing antibodies target a haemagglutinin anchor epitope.

Broadly neutralizing antibodies that target epitopes of haemagglutinin on the influenza virus have the potential to provide near universal protection against influenza virus infection1. However, viral mutants that escape broadly neutralizing antibodies have been reported2,3. The identification of broadly neutralizing antibody classes that can neutralize viral escape mutants is critical for universal influenza virus vaccine design. Here we report a distinct class of broadly neutralizing antibodies that target a discrete membrane-proximal anchor epitope of the haemagglutinin stalk domain. Anchor epitope-targeting antibodies are broadly neutralizing across H1 viruses and can cross-react with H2 and H5 viruses that are a pandemic threat. Antibodies that target this anchor epitope utilize a highly restricted repertoire, which encodes two public binding motifs that make extensive contacts with conserved residues in the fusion peptide. Moreover, anchor epitope-targeting B cells are common in the human memory B cell repertoire and were recalled in humans by an oil-in-water adjuvanted chimeric haemagglutinin vaccine4,5, which is a potential universal influenza virus vaccine. To maximize protection against seasonal and pandemic influenza viruses, vaccines should aim to boost this previously untapped source of broadly neutralizing antibodies that are widespread in the human memory B cell pool.

Germline de novo variant F747S extends the phenotypic spectrum of CACNA1D Ca2+ channelopathies.

Germline gain-of-function missense variants in the pore-forming Cav1.3 α1-subunit (CACNA1D gene) confer high risk for a severe neurodevelopmental disorder with or without endocrine symptoms. Here, we report a 4-week-old new-born with the novel de novo missense variant F747S with a so far not described prominent jittering phenotype in addition to symptoms previously reported for CACNA1D mutations including developmental delay, elevated aldosterone level and transient hypoglycemia. We confirmed the pathogenicity of this variant in whole-cell patch-clamp experiments with wild-type and F747S mutant channels heterologously expressed together with α2δ1 and cytosolic ß3 or membrane-bound ß2a subunits. Mutation F747S caused the quantitatively largest shift in the voltage dependence of activation (-28 mV) reported so far for CACNA1D germline mutations. It also shifted inactivation to more negative voltages, slowed the time course of current inactivation and slowed current deactivation upon repolarization with both co-expressed ß-subunits. In silico modelling and molecular docking, simulations revealed that this gain-of-function phenotype can be explained by formation of a novel inter-domain hydrogen bond between mutant residues S747 (IIS6) with N1145 (IIIS6) stabilizing selectively the activated open channel state. F747S displayed 2-6-fold increased sensitivity for the L-type Ca2+ channel blocker isradipine compared to wild type. Our data confirm the pathogenicity of the F747S variant with very strong gain-of-function gating changes, which may contribute to the novel jittering phenotype. Increased sensitivity for isradipine suggests this drug for potential symptomatic off-label treatment for carriers of this mutation.

Defining and Studying B Cell Receptor and TCR Interactions.

BCRs (Abs) and TCRs (or adaptive immune receptors [AIRs]) are the means by which the adaptive immune system recognizes foreign and self-antigens, playing an integral part in host defense, as well as the emergence of autoimmunity. Importantly, the interaction between AIRs and their cognate Ags defies a simple key-in-lock paradigm and is instead a complex many-to-many mapping between an individual's massively diverse AIR repertoire, and a similarly diverse antigenic space. Understanding how adaptive immunity balances specificity with epitopic coverage is a key challenge for the field, and terms such as broad specificity, cross-reactivity, and polyreactivity remain ill-defined and are used inconsistently. In this Immunology Notes and Resources article, a group of experimental, structural, and computational immunologists define commonly used terms associated with AIR binding, describe methodologies to study these binding modes, as well as highlight the implications of these different binding modes for therapeutic design.

A novel calcium channel Cavß2 splice variant with unique properties predominates in the retina.

Cavß subunits are essential for surface expression of voltage-gated calcium channel complexes and crucially modulate biophysical properties like voltage-dependent inactivation. Here, we describe the discovery and characterization of a novel Cavß2 variant with distinct features that predominates in the retina. We determined spliced exons in retinal transcripts of the Cacnb2 gene, coding for Cavß2, by RNA-Seq data analysis and quantitative PCR. We cloned a novel Cavß2 splice variant from mouse retina, which we are calling ß2i, and investigated biophysical properties of calcium currents with this variant in a heterologous expression system as well as its intrinsic membrane interaction when expressed alone. Our data showed that ß2i predominated in the retina with expression in photoreceptors and bipolar cells. Furthermore, we observed that the ß2i N-terminus exhibited an extraordinary concentration of hydrophobic residues, a distinct feature not seen in canonical variants. The biophysical properties resembled known membrane-associated variants, and ß2i exhibited both a strong membrane association and a propensity for clustering, which depended on hydrophobic residues in its N-terminus. We considered available Cavß structure data to elucidate potential mechanisms underlying the observed characteristics but resolved N-terminus structures were lacking and thus, precluded clear conclusions. With this description of a novel N-terminus variant of Cavß2, we expand the scope of functional variation through N-terminal splicing with a distinct form of membrane attachment. Further investigation of the molecular mechanisms underlying the features of ß2i could provide new angles on the way Cavß subunits modulate Ca2+ channels at the plasma membrane.

Defining the cross-reactivity between peanut allergens Ara h 2 and Ara h 6 using monoclonal antibodies.

In peanut allergy, Arachis hypogaea 2 (Ara h 2) and Arachis hypogaea 6 (Ara h 6) are two clinically relevant peanut allergens with known structural and sequence homology and demonstrated cross-reactivity. We have previously utilized X-ray crystallography and epitope binning to define the epitopes on Ara h 2. We aimed to quantitatively characterize the cross-reactivity between Ara h 2 and Ara h 6 on a molecular level using human monoclonal antibodies (mAbs) and structural characterization of allergenic epitopes. We utilized mAbs cloned from Ara h 2 positive single B cells isolated from peanut-allergic, oral immunotherapy-treated patients to quantitatively analyze cross-reactivity between recombinant Ara h 2 (rAra h 2) and Ara h 6 (rAra h 6) proteins using biolayer interferometry and indirect inhibitory ELISA. Molecular dynamics simulations assessed time-dependent motions and interactions in the antibody-antigen complexes. Three epitopes-conformational epitopes 1.1 and 3, and the sequential epitope KRELRNL/KRELMNL-are conserved between Ara h 2 and Ara h 6, while two more conformational and three sequential epitopes are not. Overall, mAb affinity was significantly lower to rAra h 6 than it was to rAra h 2. This difference in affinity was primarily due to increased dissociation of the antibodies from rAra h 6, a phenomenon explained by the higher conformational flexibility of the Ara h 6-antibody complexes in comparison to Ara h 2-antibody complexes. Our results further elucidate the cross-reactivity of peanut 2S albumins on a molecular level and support the clinical immunodominance of Ara h 2.

Cross-linking disulfide bonds govern solution structures of diabodies.

In the last years, antibodies have emerged as a promising new class of therapeutics, due to their combination of high specificity with long serum half-life and low risk of side-effects. Diabodies are a popular novel antibody format, consisting of two Fv domains connected with short linkers. Like IgG antibodies, they simultaneously bind two target proteins. However, they offer altered properties, given their smaller size and higher rigidity. In this study, we conducted the-to our knowledge-first molecular dynamics (MD) simulations of diabodies and find a surprisingly high conformational flexibility in the relative orientation of the two Fv domains. We observe rigidifying effects through the introduction of disulfide bonds in the Fv -Fv interface and characterize the effect of different disulfide bond locations on the conformation. Additionally, we compare VH -VL orientations and paratope dynamics between diabodies and an antigen binding fragment (Fab) of the same sequence. We find mostly consistent structures and dynamics, indicating similar antigen binding properties. The most significant differences can be found within the CDR-H2 loop dynamics. Of all CDR loops, the CDR-H2 is located closest to the artificial Fv -Fv interface. All examined diabodies show similar VH -VL orientations, Fv -Fv packing and CDR loop conformations. However, the variant with a P14C-K64C disulfide bond differs most from the Fab in our measures, including the CDR-H3 loop conformational ensemble. This suggests altered antigen binding properties and underlines the need for careful validation of the disulfide bond locations in diabodies.

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.

PEP-Patch: Electrostatics in Protein-Protein Recognition, Specificity, and Antibody Developability.

The electrostatic properties of proteins arise from the number and distribution of polar and charged residues. Electrostatic interactions in proteins play a critical role in numerous processes such as molecular recognition, protein solubility, viscosity, and antibody developability. Thus, characterizing and quantifying electrostatic properties of a protein are prerequisites for understanding these processes. Here, we present PEP-Patch, a tool to visualize and quantify the electrostatic potential on the protein surface in terms of surface patches, denoting separated areas of the surface with a common physical property. We highlight its applicability to elucidate protease substrate specificity and antibody-antigen recognition and predict heparin column retention times of antibodies as an indicator of pharmacokinetics.

Structural mechanism of Fab domain dissociation as a measure of interface stability.

Therapeutic antibodies should not only recognize antigens specifically, but also need to be free from developability issues, such as poor stability. Thus, the mechanistic understanding and characterization of stability are critical determinants for rational antibody design. In this study, we use molecular dynamics simulations to investigate the melting process of 16 antigen binding fragments (Fabs). We describe the Fab dissociation mechanisms, showing a separation in the VH-VL and in the CH1-CL domains. We found that the depths of the minima in the free energy curve, corresponding to the bound states, correlate with the experimentally determined melting temperatures. Additionally, we provide a detailed structural description of the dissociation mechanism and identify key interactions in the CDR loops and in the CH1-CL interface that contribute to stabilization. The dissociation of the VH-VL or CH1-CL domains can be represented by conformational changes in the bend angles between the domains. Our findings elucidate the melting process of antigen binding fragments and highlight critical residues in both the variable and constant domains, which are also strongly germline dependent. Thus, our proposed mechanisms have broad implications in the development and design of new and more stable antigen binding fragments.

Essential role of a conserved aspartate for the enzymatic activity of plasmanylethanolamine desaturase.

Plasmalogens are an abundant class of glycerophospholipids in the mammalian body, with special occurrence in the brain and in immune cell membranes. Plasmanylethanolamine desaturase (PEDS1) is the final enzyme of plasmalogen biosynthesis, which introduces the characteristic 1-O-alk-1'-enyl double bond. The recent sequence identification of PEDS1 as transmembrane protein 189 showed that its protein sequence is related to a special class of plant desaturases (FAD4), with whom it shares a motif of 8 conserved histidines, which are essential for the enzymatic activity. In the present work, we wanted to gain more insight into the sequence-function relationship of this enzyme and mutated to alanine additional 28 amino acid residues of murine plasmanylethanolamine desaturase including those 20 residues, which are also totally conserved-in addition to the eight-histidine-motif-among the animal PEDS1 and plant FAD4 plant desaturases. We measured the enzymatic activity by transient transfection of tagged murine PEDS1 expression clones to a PEDS1-deficient human HAP1 cell line by monitoring of labeled plasmalogens formed from supplemented 1-O-pyrenedecyl-sn-glycerol in relation to recombinant protein expression. Surprisingly, only a single mutation, namely aspartate 100, led to a total loss of PEDS1 activity. The second strongest impact on enzymatic activity had mutation of phenylalanine 118, leaving only 6% residual activity. A structural model obtained by homology modelling to available structures of stearoyl-CoA reductase predicted that this aspartate 100 residue interacts with histidine 96, and phenylalanine 118 interacts with histidine 187, both being essential histidines assumed to be involved in the coordination of the di-metal center of the enzyme.

Explicit solvation thermodynamics in ionic solution: extending grid inhomogeneous solvation theory to solvation free energy of salt-water mixtures.

Hydration thermodynamics play a fundamental role in fields ranging from the pharmaceutical industry to environmental research. Numerous methods exist to predict solvation thermodynamics of compounds ranging from small molecules to large biomolecules. Arguably the most precise methods are those based on molecular dynamics (MD) simulations in explicit solvent. One theory that has seen increased use is inhomogeneous solvation theory (IST). However, while many applications require accurate description of salt-water mixtures, no implementation of IST is currently able to estimate solvation properties involving more than one solvent species. Here, we present an extension to grid inhomogeneous solvation theory (GIST) that can take salt contributions into account. At the example of carbazole in 1 M NaCl solution, we compute the solvation energy as well as first and second order entropies. While the effect of the first order ion entropy is small, both the water-water and water-ion entropies contribute strongly. We show that the water-ion entropies are efficiently approximated using the Kirkwood superposition approximation. However, this approach cannot be applied to the water-water entropy. Furthermore, we test the quantitative validity of our method by computing salting-out coefficients and comparing them to experimental data. We find a good correlation to experimental salting-out constants, while the absolute values are overpredicted due to the approximate second order entropy. Since ions are frequently used in MD, either to neutralize the system or as a part of the investigated process, our method greatly extends the applicability of GIST. The use-cases range from biopharmaceuticals, where many assays require high salt concentrations, to environmental research, where solubility in sea water is important to model the fate of organic substances.

CACNA1I gain-of-function mutations differentially affect channel gating and cause neurodevelopmental disorders.

T-type calcium channels (Cav3.1 to Cav3.3) regulate low-threshold calcium spikes, burst firing and rhythmic oscillations of neurons and are involved in sensory processing, sleep, and hormone and neurotransmitter release. Here, we examined four heterozygous missense variants in CACNA1I, encoding the Cav3.3 channel, in patients with variable neurodevelopmental phenotypes. The p.(Ile860Met) variant, affecting a residue in the putative channel gate at the cytoplasmic end of the IIS6 segment, was identified in three family members with variable cognitive impairment. The de novo p.(Ile860Asn) variant, changing the same amino acid residue, was detected in a patient with severe developmental delay and seizures. In two additional individuals with global developmental delay, hypotonia, and epilepsy, the variants p.(Ile1306Thr) and p.(Met1425Ile), substituting residues at the cytoplasmic ends of IIIS5 and IIIS6, respectively, were found. Because structure modelling indicated that the amino acid substitutions differentially affect the mobility of the channel gate, we analysed possible effects on Cav3.3 channel function using patch-clamp analysis in HEK293T cells. The mutations resulted in slowed kinetics of current activation, inactivation, and deactivation, and in hyperpolarizing shifts of the voltage-dependence of activation and inactivation, with Cav3.3-I860N showing the strongest and Cav3.3-I860M the weakest effect. Structure modelling suggests that by introducing stabilizing hydrogen bonds the mutations slow the kinetics of the channel gate and cause the gain-of-function effect in Cav3.3 channels. The gating defects left-shifted and increased the window currents, resulting in increased calcium influx during repetitive action potentials and even at resting membrane potentials. Thus, calcium toxicity in neurons expressing the Cav3.3 variants is one likely cause of the neurodevelopmental phenotype. Computer modelling of thalamic reticular nuclei neurons indicated that the altered gating properties of the Cav3.3 disease variants lower the threshold and increase the duration and frequency of action potential firing. Expressing the Cav3.3-I860N/M mutants in mouse chromaffin cells shifted the mode of firing from low-threshold spikes and rebound burst firing with wild-type Cav3.3 to slow oscillations with Cav3.3-I860N and an intermediate firing mode with Cav3.3-I860M, respectively. Such neuronal hyper-excitability could explain seizures in the patient with the p.(Ile860Asn) mutation. Thus, our study implicates CACNA1I gain-of-function mutations in neurodevelopmental disorders, with a phenotypic spectrum ranging from borderline intellectual functioning to a severe neurodevelopmental disorder with epilepsy.

Grid inhomogeneous solvation theory for cross-solvation in rigid solvents.

Grid Inhomogeneous Solvation Theory (GIST) has proven useful to calculate localized thermodynamic properties of water around a solute. Numerous studies have leveraged this information to enhance structure-based binding predictions. We have recently extended GIST toward chloroform as a solvent to allow the prediction of passive membrane permeability. Here, we further generalize the GIST algorithm toward all solvents that can be modeled as rigid molecules. This restriction is inherent to the method and is already present in the inhomogeneous solvation theory. Here, we show that our approach can be applied to various solvent molecules by comparing the results of GIST simulations with thermodynamic integration (TI) calculations and experimental results. Additionally, we analyze and compare a matrix consisting of 100 entries of ten different solvent molecules solvated within each other. We find that the GIST results are highly correlated with TI calculations as well as experiments. For some solvents, we find Pearson correlations of up to 0.99 to the true entropy, while others are affected by the first-order approximation more strongly. The enthalpy-entropy splitting provided by GIST allows us to extend a recently published approach, which estimates higher order entropies by a linear scaling of the first-order entropy, to solvents other than water. Furthermore, we investigate the convergence of GIST in different solvents. We conclude that our extension to GIST reliably calculates localized thermodynamic properties for different solvents and thereby significantly extends the applicability of this widely used method.

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.

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.

Conformational Ensembles of Antibodies Determine Their Hydrophobicity.

A major challenge in the development of antibody biotherapeutics is their tendency to aggregate. One root cause for aggregation is exposure of hydrophobic surface regions to the solvent. Many current techniques predict the relative aggregation propensity of antibodies via precalculated scales for the hydrophobicity or aggregation propensity of single amino acids. However, those scales cannot describe the nonadditive effects of a residue's surrounding on its hydrophobicity. Therefore, they are inherently limited in their ability to describe the impact of subtle differences in molecular structure on the overall hydrophobicity. Here, we introduce a physics-based approach to describe hydrophobicity in terms of the hydration free energy using grid inhomogeneous solvation theory (GIST). We apply this method to assess the effects of starting structures, conformational sampling, and protonation states on the hydrophobicity of antibodies. Our results reveal that high-quality starting structures, i.e., crystal structures, are crucial for the prediction of hydrophobicity and that conformational sampling can compensate errors introduced by the starting structure. On the other hand, sampling of protonation states only leads to good results when combined with high-quality structures, whereas it can even be detrimental otherwise. We conclude by pointing out that a single static homology model may not be adequate for predicting hydrophobicity.

Ion-pair interactions between voltage-sensing domain IV and pore domain I regulate CaV1.1 gating.

The voltage-gated calcium channel CaV1.1 belongs to the family of pseudo-heterotetrameric cation channels, which are built of four structurally and functionally distinct voltage-sensing domains (VSDs) arranged around a common channel pore. Upon depolarization, positive gating charges in the S4 helices of each VSD are moved across the membrane electric field, thus generating the conformational change that prompts channel opening. This sliding helix mechanism is aided by the transient formation of ion-pair interactions with countercharges located in the S2 and S3 helices within the VSDs. Recently, we identified a domain-specific ion-pair partner of R1 and R2 in VSD IV of CaV1.1 that stabilizes the activated state of this VSD and regulates the voltage dependence of current activation in a splicing-dependent manner. Structure modeling of the entire CaV1.1 in a membrane environment now revealed the participation in this process of an additional putative ion-pair partner (E216) located outside VSD IV, in the pore domain of the first repeat (IS5). This interdomain interaction is specific for CaV1.1 and CaV1.2 L-type calcium channels. Moreover, in CaV1.1 it is sensitive to insertion of the 19 amino acid peptide encoded by exon 29. Whole-cell patch-clamp recordings in dysgenic myotubes reconstituted with wild-type or E216 mutants of GFP-CaV1.1e (lacking exon 29) showed that charge neutralization (E216Q) or removal of the side chain (E216A) significantly shifted the voltage dependence of activation (V1/2) to more positive potentials, suggesting that E216 stabilizes the activated state. Insertion of exon 29 in the GFP-CaV1.1a splice variant strongly reduced the ionic interactions with R1 and R2 and caused a substantial right shift of V1/2, whereas no further shift of V1/2 was observed on substitution of E216 with A or Q. Together with our previous findings, these results demonstrate that inter- and intradomain ion-pair interactions cooperate in the molecular mechanism regulating VSD function and channel gating in CaV1.1.

Cav1.4 dysfunction and congenital stationary night blindness type 2.

Cav1.4 L-type Ca2+ channels are predominantly expressed in retinal neurons, particularly at the photoreceptor terminals where they mediate sustained Ca2+ entry needed for continuous neurotransmitter release at their ribbon synapses. Cav1.4 channel gating properties are controlled by accessory subunits, associated regulatory proteins, and also alternative splicing. In humans, mutations in the CACNA1F gene encoding for Cav1.4 channels are associated with X-linked retinal disorders such as congenital stationary night blindness type 2. Mutations in the Cav1.4 protein result in a spectrum of altered functional channel activity. Several mouse models broadened our understanding of the role of Cav1.4 channels not only as Ca2+ source at retinal synapses but also as synaptic organizers. In this review, we highlight different structural and functional phenotypes of Cav1.4 mutations that might also occur in patients with congenital stationary night blindness type 2. A further important yet mostly neglected aspect that we discuss is the influence of alternative splicing on channel dysfunction. We conclude that currently available functional phenotyping strategies should be refined and summarize potential specific therapeutic options for patients carrying Cav1.4 mutations. Importantly, the development of new therapeutic approaches will permit a deeper understanding of not only the disease pathophysiology but also the physiological function of Cav1.4 channels in the retina.

VH -VL interdomain dynamics observed by computer simulations and NMR.

The relative orientation of the two variable domains, VH and VL , influences the shape of the antigen binding site, that is, the paratope, and is essential to understand antigen specificity. ABangle characterizes the VH -VL orientation by using five angles and a distance and compares it to other known structures. Molecular dynamics simulations of antibody variable domains (Fvs) reveal fluctuations in the relative domain orientations. The observed dynamics between these domains are confirmed by NMR experiments on a single-chain variable fragment antibody (scFv) in complex with IL-1ß and an antigen-binding fragment (Fab). The variability of these relative domain orientations can be interpreted as a structural feature of antibodies, which increases the antibody repertoire significantly and can enlarge the number of possible binding partners substantially. The movements of the VH and VL domains are well sampled with molecular dynamics simulations and are in agreement with the NMR ensemble. Fast Fourier transformation of the ABangle metrics allows to assign timescales of 0.1-10 GHz to the fastest collective interdomain movements. The results clearly show the necessity of dynamics to understand and characterize the favorable orientations of the VH and VL domains implying a considerable binding interface flexibility and reveal in all antibody fragments (Fab, scFv, and Fv) very similar VH -VL interdomain variations comparable to the distributions observed for known X-ray structures of antibodies. SIGNIFICANCE STATEMENT: Antibodies have become key players as therapeutic agents. The binding ability of antibodies is determined by the antigen-binding fragment (Fab), in particular the variable fragment region (Fv). Antigen-binding is mediated by the complementarity-determining regions consisting of six loops, each three of the heavy and light chain variable domain VH and VL . The relative orientation of the VH and VL domains influences the shape of the antigen-binding site and is a major objective in antibody design. In agreement with NMR experiments and molecular dynamics simulations, we show a considerable binding site flexibility in the low nanosecond timescale. Thus we suggest that this flexibility and its implications for binding and specificity should be considered when designing and optimizing therapeutic antibodies.