Decoupling peptide binding from T cell receptor recognition with engineered chimeric MHC-I molecules

Major Histocompatibility Complex class I (MHC-I) molecules display self, viral or aberrant epitopic peptides to T cell receptors (TCRs), which employ interactions between complementarity-determining regions with both peptide and MHC-I heavy chain ‘framework’ residues to recognize specific Human Leucocyte Antigens (HLAs). The highly polymorphic nature of the HLA peptide-binding groove suggests a malleability of interactions within a common structural scaffold. Here, using structural data from peptide:MHC-I and pMHC:TCR structures, we first identify residues important for peptide and/or TCR binding. We then outline a fixed-backbone computational design approach for engineering synthetic molecules that combine peptide binding and TCR recognition surfaces from existing HLA allotypes. X-ray crystallography demonstrates that chimeric molecules bridging divergent HLA alleles can bind selected peptide antigens in a specified backbone conformation. Finally, in vitro tetramer staining and biophysical binding experiments using chimeric pMHC-I molecules presenting established antigens further demonstrate the requirement of TCR recognition on interactions with HLA framework residues, as opposed to interactions with peptide-centric Chimeric Antigen Receptors (CARs). Our results underscore a novel, structure-guided platform for developing synthetic HLA molecules with desired properties as screening probes for peptide-centric interactions with TCRs and other therapeutic modalities

Role of water in Protein Aggregation and Amyloid Polymorphism

A variety of neurodegenerative diseases are associated with the formation of amyloid plaques. Our incomplete understanding of this process underscores the need to decipher the principles governing protein aggregation. Most experimental and simulation studies have been interpreted largely from the perspective of proteins: the role of solvent has been relatively overlooked. In this Account, we provide a perspective on how interactions with water affect folding landscapes of A$\beta$ monomers, A$\beta_{16-22}$ oligomer formation, and protofilament formation in a Sup35 peptide. Simulations show that the formation of aggregation-prone structures (N$^*$) similar to the structure in the fibril requires overcoming high desolvation barrier. The mechanism of protofilament formation in a polar Sup35 peptide fragment illustrates that water dramatically slows down self-assembly. Release of water trapped in the pores as water wires creates protofilament with a dry interface. Similarly, one of the main driving force for addition of a solvated monomer to a preformed fibril is the entropy gain of released water. We conclude by postulating that two-step model for protein crystallization must also hold for higher order amyloid structure formation starting from N$^*$. Multiple N$^*$ structures with varying water content results in a number of distinct water-laden polymorphic structures. In predominantly hydrophobic sequences, water accelerates fibril formation. In contrast, water-stabilized metastable intermediates dramatically slow down fibril growth rates in hydrophilic sequences.Comment: 27 pages, 4 figures; Accounts of Chemical Research, 201

Calorimetric Investigation of Copper Binding in the N-Terminal Region of the Prion Protein at Low Copper Loading: Evidence for an Entropically Favorable First Binding Event

Although the Cu²⁺-binding sites of the prion protein have been well studied when the protein is fully saturated by Cu²⁺, the Cu²⁺-loading mechanism is just beginning to come into view. Because the Cu²⁺-binding modes at low and intermediate Cu²⁺ occupancy necessarily represent the highest-affinity binding modes, these are very likely populated under physiological conditions, and it is thus essential to characterize them in order to understand better the biological function of copper–prion interactions. Besides binding-affinity data, almost no other thermodynamic parameters (e.g., Δ<i>H</i> and Δ<i>S</i>) have been measured, thus leaving undetermined the enthalpic and entropic factors that govern the free energy of Cu²⁺ binding to the prion protein. In this study, isothermal titration calorimetry (ITC) was used to quantify the thermodynamic parameters (<i>K</i>, Δ<i>G</i>, Δ<i>H</i>, and <i>T</i>Δ<i>S</i>) of Cu²⁺ binding to a peptide, PrP­(23–28, 57–98), that encompasses the majority of the residues implicated in Cu²⁺ binding by full-length PrP. Use of the buffer <i>N</i>-(2-acetomido)-aminoethanesulfonic acid (ACES), which is also a well-characterized Cu²⁺ chelator, allowed for the isolation of the two highest affinity binding events. Circular dichroism spectroscopy was used to characterize the different binding modes as a function of added Cu²⁺. The <i>K</i>_d values determined by ITC, 7 and 380 nM, are well in line with those reported by others. The first binding event benefits significantly from a positive entropy, whereas the second binding event is enthalpically driven. The thermodynamic values associated with Cu²⁺ binding by the Aβ peptide, which is implicated in Alzheimer’s disease, bear striking parallels to those found here for the prion protein

Dimer Formation Enhances Structural Differences between Amyloid β-Protein (1–40) and (1–42): An Explicit-Solvent Molecular Dynamics Study

Amyloid -protein (A) is central to the pathology of Alzheimer's disease. A 5% difference in the primary structure of the two predominant alloforms, A and A, results in distinct assembly pathways and toxicity properties. Discrete molecular dynamics (DMD) studies of A and A assembly resulted in alloform-specific oligomer size distributions consistent with experimental findings. Here, a large ensemble of DMD–derived A and A monomers and dimers was subjected to fully atomistic molecular dynamics (MD) simulations using the OPLS-AA force field combined with two water models, SPCE and TIP3P. The resulting all-atom conformations were slightly larger, less compact, had similar turn and lower -strand propensities than those predicted by DMD. Fully atomistic A and A monomers populated qualitatively similar free energy landscapes. In contrast, the free energy landscape of A dimers indicated a larger conformational variability in comparison to that of A dimers. A dimers were characterized by an increased flexibility in the N-terminal region D1-R5 and a larger solvent exposure of charged amino acids relative to A dimers. Of the three positively charged amino acids, R5 was the most and K16 the least involved in salt bridge formation. This result was independent of the water model, alloform, and assembly state. Overall, salt bridge propensities increased upon dimer formation. An exception was the salt bridge propensity of K28, which decreased upon formation of A dimers and was significantly lower than in A dimers. The potential relevance of the three positively charged amino acids in mediating the A oligomer toxicity is discussed in the light of available experimental data

Conformational Preferences of a 14-Residue Fibrillogenic Peptide from Acetylcholinesterase†

A 14-residue fragment from near the C-terminus of the enzyme acetylcholinesterase (AChE) is believed to have a neurotoxic/neurotrophic effect acting via an unknown pathway. While the peptide is α-helical in the full-length enzyme, the structure and association mechanism of the fragment are unknown. Using multiple molecular dynamics simulations, starting from a tetrameric complex of the association domain of AChE and systematicall disassembled subsets that include the peptide fragment, we show that the fragment is incapable of retaining its helicity in solution. Extensive replica exchange Monte Carlo folding and unfolding simulations in implicit solvent with capped and uncappted termini failed to converge to any consistent cluster of structures, suggesting that the fragment remains largely unstructured in solution under the conditions considered. Furthermore, extended molecular dynamics simulations of two steric zipper models show that the peptide is likely to form a zipper with antiparallel sheets and that peptides with mutations known to prevent fibril formation likely do so by interfering with this packing. The results demonstrate how the local environment of a peptide can stabilize a particular conformation

2017 update of the WSES guidelines for emergency repair of complicated abdominal wall hernias

Emergency repair of complicated abdominal wall hernias may be associated with worsen outcome and a significant rate of postoperative complications. There is no consensus on management of complicated abdominal hernias. The main matter of debate is about the use of mesh in case of intestinal resection and the type of mesh to be used. Wound infection is the most common complication encountered and represents an immense burden especially in the presence of a mesh. The recurrence rate is an important topic that influences the final outcome. A World Society of Emergency Surgery (WSES) Consensus Conference was held in Bergamo in July 2013 with the aim to define recommendations for emergency repair of abdominal wall hernias in adults. This document represents the executive summary of the consensus conference approved by a WSES expert panel. In 2016, the guidelines have been revised and updated according to the most recent available literature.Peer reviewe

WSES guidelines for emergency repair of complicated abdominal wall hernias

Peer reviewe

Computational methods for the prediction of GPCRs coupling selectivity

GPCRs comprise a wide and diverse class of eukaryotic transmembrane proteins with well-established pharmacological significance. As a consequence of recent genome projects, there is a wealth of information at the sequence level that lacks any functional annotation. These receptors, often quoted as orphan GPCRs, could potentially lead to novel drug targets. However, typical experiments that aim at elucidating their function are hampered by the lack of knowledge on their selective coupling partners at the interior of the cell, the G-proteins. Up-to-date, computational efforts to predict properties of GPCRs have been focused mainly on the ligand-binding specificity, while the aspect of coupling has been less studied. Here, we present the main motivations, drawbacks, and results from the application of bioinformatics techniques to predict the coupling specificity of GPCRs to G-proteins, and discuss the application of the most successful methods in both experimental works that focus on a single receptor and large-scale genome annotation studies. © 2009, IGI Global