Deciphering deamidation and isomerization in therapeutic proteins: Effect of neighboring residue.

Deamidation of asparagine (Asn) and isomerization of aspartic acid (Asp) residues are among the most commonly observed spontaneous post-translational modifications (PTMs) in proteins. Understanding and predicting a protein sequence's propensity for such PTMs can help expedite protein therapeutic discovery and development. In this study, we used proton-affinity calculations with semi-empirical quantum mechanics and microsecond long equilibrium molecular dynamics simulations to investigate mechanistic roles of structural conformation and chemical environment in dictating spontaneous degradation of Asn and Asp residues in 131 clinical-stage therapeutic antibodies. Backbone secondary structure, side-chain rotamer conformation and solvent accessibility were found to be key molecular indicators of Asp isomerization and Asn deamidation. Comparative analysis of backbone dihedral angles along with N-H proton affinity calculations provides a mechanistic explanation for the strong influence of the identity of the n + 1 residue on the rate of Asn/Asp degradation. With these findings, we propose a minimalistic physics-based classification model that can be leveraged to predict deamidation and isomerization propensity of proteins.

Rational identification and characterisation of peptide ligands for targeting polysialic acid.

The alpha-2,8-linked form of the polysaccharide polysialic acid (PSA) has widespread implications in physiological and pathological processes, ranging from neurological development to disease progression. Though the high electronegativity and excluded volume of PSA often promotes interference of biomolecular interactions, PSA-binding ligands have important implications for both biological processes and biotechnological applications. As such, the design, identification, and characterisation of novel ligands towards PSA is critical for expanding knowledge of PSA interactions and achieving selective glycan targeting. Here, we report on a rational approach for the identification of alpha-2,8-PSA-binding peptides, involving design from the endogenous ligand Siglec-11 and multi-platform characterisation of peptide binding. Microarray-based examination of peptides revealed charge and sequence characteristics influencing peptide affinity to PSA, and carbohydrate-peptide binding was further quantified with a novel fluorescence anisotropy assay. PSA-binding peptides exhibited specific binding to polymeric SA, as well as different degrees of selective binding in various conditions, including competition with PSA of alternating 2,8/9-linkages and screening with PSA-expressing cells. A computational study of Siglec-11 and Siglec-11-derived peptides offered synergistic insight into ligand binding. These results demonstrate the potential of PSA-binding peptides for selective targeting and highlight the importance of the approaches described herein for the study of carbohydrate interactions.

Paracellular Gatekeeping: What Does It Take for an Ion to Pass Through a Tight Junction Pore?

Tight junction pores are physiological gatekeepers of paracellular transport in epithelial tissues. Conventionally, tight junction permeability is determined via in vitro electrophysiology measurements; however, the macroscopic readout does not provide molecular-level understanding into the mechanism of ion permeation. Insight into the factors governing selectivity across the paracellular space is just emerging. In this study, we investigated tight junction pores comprising of claudin-2 and claudin-5 proteins that are structurally similar to subnanometer radii but have measurably different in vitro ion permeabilities. To evaluate the mechanistic differences in ion transport across the pores, we computed the free-energy profiles and relative rate constants for the transport of monovalent (Na+, K+, Cl-) and divalent (Mg2+ and Ca2+) ions through the pores using replica exchange metadynamics. In claudin-2, we demonstrate how a single residue dictates selective permeability of Na+ and K+ ions. In claudin-5, we found no clear preference for anion or cation selectivity; thus, pores formed by claudin-5 are indeed barriers to ion permeation. Mutations to claudin-5 that widen the pore's steric radius did not significantly impact pore selectivity, indicating that electrostatics dominate pore selectivity. The key takeaways from this work are as follows: (a) two pores that are similar in diameter and length can have dissimilar ion conductance, (b) existence of a physical pore does not guarantee ion permeability, and (c) the electrostatic environment created by the pore-lining residues dictates the ion conductivity. These mechanistic understandings of the tight junction pores are critical for the interpretation of tight junction physiology.

Computational Nanoscopy of Tight Junctions at the Blood-Brain Barrier Interface.

The selectivity of the blood-brain barrier (BBB) is primarily maintained by tight junctions (TJs), which act as gatekeepers of the paracellular space by blocking blood-borne toxins, drugs, and pathogens from entering the brain. The BBB presents a significant challenge in designing neurotherapeutics, so a comprehensive understanding of the TJ architecture can aid in the design of novel therapeutics. Unraveling the intricacies of TJs with conventional experimental techniques alone is challenging, but recently developed computational tools can provide a valuable molecular-level understanding of TJ architecture. We employed the computational methods toolkit to investigate claudin-5, a highly expressed TJ protein at the BBB interface. Our approach started with the prediction of claudin-5 structure, evaluation of stable dimer conformations and nanoscale assemblies, followed by the impact of lipid environments, and posttranslational modifications on these claudin-5 assemblies. These led to the study of TJ pores and barriers and finally understanding of ion and small molecule transport through the TJs. Some of these in silico, molecular-level findings, will need to be corroborated by future experiments. The resulting understanding can be advantageous towards the eventual goal of drug delivery across the BBB. This review provides key insights gleaned from a series of state-of-the-art nanoscale simulations (or computational nanoscopy studies) performed on the TJ architecture.

The ghrelin O-acyltransferase structure reveals a catalytic channel for transmembrane hormone acylation.

Integral membrane proteins represent a large and diverse portion of the proteome and are often recalcitrant to purification, impeding studies essential for understanding protein structure and function. By combining co-evolutionary constraints and computational modeling with biochemical validation through site-directed mutagenesis and enzyme activity assays, we demonstrate here a synergistic approach to structurally model purification-resistant topologically complex integral membrane proteins. We report the first structural model of a eukaryotic membrane-bound O-acyltransferase (MBOAT), ghrelin O-acyltransferase (GOAT), which modifies the metabolism-regulating hormone ghrelin. Our structure, generated in the absence of any experimental structural data, revealed an unanticipated strategy for transmembrane protein acylation with catalysis occurring in an internal channel connecting the endoplasmic reticulum lumen and cytoplasm. This finding validated the power of our approach to generate predictive structural models for other experimentally challenging integral membrane proteins. Our results illuminate novel aspects of membrane protein function and represent key steps for advancing structure-guided inhibitor design to target therapeutically important but experimentally intractable membrane proteins.

Palmitoylation of Claudin-5 Proteins Influences Their Lipid Domain Affinity and Tight Junction Assembly at the Blood-Brain Barrier Interface.

Post-translational lipid modification of integral membrane proteins is recognized as a key mechanism to modulate protein-protein and membrane-protein associations. Despite numerous reports of lipid-modified proteins, molecular-level understanding of the influence of lipid-modification of key membrane proteins remains elusive. This study focuses on the lipid modification of one such protein-claudin-5, a critical component of the blood-brain barrier tight junctions. Claudin-5 proteins are responsible for regulating the size and charge-selective permeability at the blood-brain interface. Palmitoylation of the claudin family of proteins is implicated in influencing the tight junction permeability in prior experimental studies. Here, we investigate the impact of palmitoylation on claudin-5 self-assembly using multiscale molecular simulations. To elucidate protein-membrane interactions, we used three model membrane compositions (endoplasmic reticulum, cholesterol-enriched endoplasmic reticulum, and plasma membrane) that mimic the complexity of cell organelles encountered by a typical membrane protein in its secretion pathway. The results show that palmitoylation enhances protein's affinity for cholesterol-rich domains in a membrane, and it can elicit a site-specific response based on the location of the palmitoyl chain on the protein. Also, in claudin-5 self-assembly, palmitoylation restricts specific protein-protein conformations. Overall, this study demonstrates the significance of post-translational lipid modification of proteins in cellular and subcellular membranes, and the impact palmitoylation can have on critical cellular functions of the protein.

Self-Assembly Simulations of Classic Claudins-Insights into the Pore Structure, Selectivity, and Higher Order Complexes.

Tight junction (TJ) protein assembly controls permeability across epithelial and endothelial cells; thus, biochemical interactions that control the TJ assembly have physiological and biomedical significance. In this work, we employed multiscale simulations to probe the TJ self-assembly of five classic claudins (-1, -2, -4, -15, and -19). Claudin proteins assembled into dimeric and occasionally trimeric interfaces that subsequently formed larger polymeric strands. Using orientation-angle analysis to decompose polymeric strands, we found that individual claudins prefer certain dimer interfaces to others. Despite variations in the exact dimer populations observed in individual claudins, there appears to be an overall conformational uniformity in the type of dimeric interactions formed by the claudin family of proteins. A detailed structural characterization of the trimeric assemblies revealed that they could be putative receptors for trimeric Clostridium perfringens enterotoxin. Full characterization of the claudin-2 dimer interface revealed a cysteine cross-linkable interaction, which could be assembled into a symmetric pore of 7.4 Å average diameter. We extended the analysis of pore structure to other classic claudins and found that the distribution of polar residues lining the pore volume varied considerably between the barrier- and pore-forming claudins, potentially delineating the functionality in classic claudins.

Architecture of the paracellular channels formed by claudins of the blood-brain barrier tight junctions.

Tight junctions (TJs) are key players in determining tissue-specific paracellular permeability across epithelial and endothelial membranes. Claudin proteins, the primary determinants of TJs structure and functionality, assemble in paracellular spaces to form channels and pores that are charge and size selective. Here, using molecular dynamics (MD) simulations, we elucidate the molecular assembly of claudin-3 and claudin-5 proteins of blood-brain barrier TJs. Despite having a high degree of sequence and structural similarity, these two claudins form different types of cis-interactions. Molecular docking of the observed cis-interfaces into trans-forms revealed two putative pore models that were also observed in the self-assembly simulations. The observed pore structures (pore I and II) have pore-lining residues that have been previously reported in the literature. The pore I model is consistent with a previously reported claudin-15 model. The pore II model, also consistent with biochemical results, has not been reported previously. Further analysis using in silico site-directed mutations provide convincing support for the validity of the pore II model. Using steered MD and umbrella sampling, we computed the transport properties of water and α-d-glucose through pore II. The study offers new insight into the selectivity of blood-brain barrier TJs.

Molecular Architecture of the Blood Brain Barrier Tight Junction Proteins--A Synergistic Computational and In Vitro Approach.

The blood-brain barrier (BBB) constituted by claudin-5 tight junctions is critical in maintaining the homeostasis of the central nervous system, but this highly selective molecular interface is an impediment for therapeutic interventions in neurodegenerative and neurological diseases. Therapeutic strategies that can exploit the paracellular transport remain elusive due to lack of molecular insights of the tight junction assembly. This study focuses on analyzing the membrane driven cis interactions of claudin-5 proteins in the formation of the BBB tight junctions. We have adopted a synergistic approach employing in silico multiscale dynamics and in vitro cross-linking experiments to study the claudin-5 interactions. Long time scale simulations of claudin-5 monomers, in seven different lipid compositions, show formation of cis dimers that subsequently aggregate into strands. In vitro formaldehyde cross-linking studies also conclusively show that cis-interacting claudin-5 dimers cross-link with short methylene spacers. Using this synergistic approach, we have identified five unique dimer interfaces in our simulations that correlate with the cross-linking experiments, four of which are mediated by transmembrane (TM) helices and the other mediated by extracellular loops (ECL). Potential of mean force calculations of these five dimers revealed that the TM mediated interfaces, which can have distinctive leucine zipper interactions in some cases, are more stable than the ECL mediated interface. Additionally, simulations show that claudin-5 dimerization is significantly influenced by the lipid microenvironment. This study captures the fundamental interactions responsible for the BBB tight junction assembly and offers a framework for extending this work to other tight junctions found in the body.

Simulating Gram-Negative Bacterial Outer Membrane: A Coarse Grain Model.

The cell envelope of Gram-negative bacteria contains a lipopolysaccharide (LPS) rich outer membrane that acts as the first line of defense for bacterial cells in adverse physical and chemical environments. The LPS macromolecule has a negatively charged oligosaccharide domain that acts as an ionic brush, limiting the permeability of charged chemical agents through the membrane. Besides the LPS, the outer membrane has radially extending O-antigen polysaccharide chains and ß-barrel membrane proteins that make the bacterial membrane physiologically unique compared to phospholipid cell membranes. Elucidating the interplay of these contributing macromolecular components and their role in the integrity of the bacterial outer membrane remains a challenge. To bridge the gap in our current understanding of the Gram-negative bacterial membrane, we have developed a coarse grained force field for outer membrane that is computationally affordable for simulating dynamical process over physiologically relevant time scales. The force field was benchmarked against available experimental and atomistic simulations data for properties such as membrane thickness, density profiles of the residues, area per lipid, gel to liquid-crystalline phase transition temperatures, and order parameters. More than 17 membrane compositions were studied with a combined simulation time of over 100 µs. A comparison of simulated structural and dynamical properties with corresponding experimental data shows that the developed force field reproduces the overall physiology of LPS rich membranes. The affordability of the developed model for long time scale simulations can be instrumental in determining the mechanistic aspects of the antimicrobial action of chemical agents as well as assist in designing antimicrobial peptides with enhanced outer membrane permeation properties.

Optical Signature of Formation of Protein Corona in the Firefly Luciferase-CdSe Quantum Dot Complex.

Theoretical investigation of protein corona is challenging because of the size of the protein-dot complex. In this work, we have addressed this computational bottleneck by combining pseudopotential + explicitly correlated Hartree-Fock QM calculations with molecular mechanics, molecular dynamics, and Monte Carlo techniques. The optical gap of a 5 nm CdSe quantum dot (Cd1159Se1183) was computed by sequential addition of luciferase (Lu), and a red shift of 8 nm in the λmax of protein corona (CdSe-Lu7) was observed.