α-Hydrazino Acid Insertion Governs Peptide Organization in Solution by Local Structure Ordering.

In this work, we have applied the concept of α-hydrazino acid insertion in a peptide sequence as a means of structurally organizing a potential protein-protein interactions (PPI) inhibitor. Hydrazino peptides characterized by the incorporation of an α-hydrazino acid at specific positions introduce an additional nitrogen atom into their backbone. This modification leads to a change in the electrostatic properties of the peptide and induces the restructuring of its hydrogen bonding network, resulting in conformational changes toward more stable structural motifs. Despite the successful use of synthetic hydrazino oligomers in binding to nucleic acids, the structural changes due to the incorporation of α-hydrazino acid into short natural peptides in solution are still poorly understood. Based on NMR data, we report structural models of p53-derived hydrazino peptides with elements of localized peptide structuring in the form of an α-, ß-, or γ-turn as a result of hydrazino modification in the peptide backbone. The modifications could potentially lead to the preorganization of a helical secondary peptide structure in a solution that is favorable for binding to a biological receptor. Spectroscopically, we observed that the ensemble averaged rapidly interconverting conformations, including isomerization of the E-Z hydrazide bond. This further increases the adaptability by expanding the conformational space of hydrazine peptides as potential protein-protein interaction antagonists.

An anti-HER2 nanobody binds to its antigen HER2 via two independent paratopes.

High-resolution structural data of complexes between antibodies and membrane receptors still represent a demanding task. In this study, we used complementary sets of experimental data to obtain a structural model of the complex formed by the human epidermal growth factor receptor 2 (HER2) and its specific nanobody A10. First we identified by NMR the residues that bind or rearrange as a consequence of the complex formation. In parallel, the complex was cross-linked, digested and the resulting peptides were characterized by mass-spectrometry to define maximal distance restraints between HER2 and A10 amino acids in their complex. These independent datasets guided a docking process, refined by molecular dynamics simulations, to develop a model of the complex and estimate per-residue free-energy contributions. Such a model explains the experimental data and identifies a second, non-canonical paratope, located in the region opposite to the conventional nanobody paratope, formed by the hypervariable loop regions LH1 and LH3. Both paratopes contributed substantially to the overall affinity by binding to independent HER2 epitopes. Nanobody mutants with substitution of key interaction residues, as indicated by the model, possess significantly lower affinity for HER2. This is the first described case of a "natural" biparatopic nanobody, directly selected by in-vitro panning.

Structure, self-assembly, and properties of a truncated reflectin variant.

Naturally occurring and recombinant protein-based materials are frequently employed for the study of fundamental biological processes and are often leveraged for applications in areas as diverse as electronics, optics, bioengineering, medicine, and even fashion. Within this context, unique structural proteins known as reflectins have recently attracted substantial attention due to their key roles in the fascinating color-changing capabilities of cephalopods and their technological potential as biophotonic and bioelectronic materials. However, progress toward understanding reflectins has been hindered by their atypical aromatic and charged residue-enriched sequences, extreme sensitivities to subtle changes in environmental conditions, and well-known propensities for aggregation. Herein, we elucidate the structure of a reflectin variant at the molecular level, demonstrate a straightforward mechanical agitation-based methodology for controlling this variant's hierarchical assembly, and establish a direct correlation between the protein's structural characteristics and intrinsic optical properties. Altogether, our findings address multiple challenges associated with the development of reflectins as materials, furnish molecular-level insight into the mechanistic underpinnings of cephalopod skin cells' color-changing functionalities, and may inform new research directions across biochemistry, cellular biology, bioengineering, and optics.

Unique Structural Features of Mule Deer Prion Protein Provide Insights into Chronic Wasting Disease.

Chronic wasting disease (CWD) is a highly infectious prion disease of cervids. Accumulation of prions, the disease-specific structural conformers of the cellular prion protein (PrPC), in the central nervous system, is the key pathological event of the disorder. The analysis of cervid PrPC sequences revealed the existence of polymorphism at position 226, in which deer PrP contains glutamine (Q), whereas elk PrP contains glutamate (E). The effects of this polymorphism on CWD are still unknown. We determined the high-resolution nuclear magnetic resonance structure of the mule deer prion protein that was compared to previously published PrP structures of elk and white-tailed deer. We found that the polymorphism Q226E could influence the long-range intramolecular interactions and packing of the ß2-α2 loop and the C-terminus of the α3 helix of cervid PrP structures. This solvent-accessible epitope is believed to be involved in prion conversion. Additional differences were observed at the beginning of the well-defined C-terminus domain, in the α2-α3 region, and in its interactions with the α1 helix. Here, we highlight the importance of the PrP structure in prion susceptibility and how single amino acid differences might influence the overall protein folding.

Enabling adoption of 2D-NMR for the higher order structure assessment of monoclonal antibody therapeutics.

The increased interest in using monoclonal antibodies (mAbs) as a platform for biopharmaceuticals has led to the need for new analytical techniques that can precisely assess physicochemical properties of these large and very complex drugs for the purpose of correctly identifying quality attributes (QA). One QA, higher order structure (HOS), is unique to biopharmaceuticals and essential for establishing consistency in biopharmaceutical manufacturing, detecting process-related variations from manufacturing changes and establishing comparability between biologic products. To address this measurement challenge, two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) methods were introduced that allow for the precise atomic-level comparison of the HOS between two proteins, including mAbs. Here, an inter-laboratory comparison involving 26 industrial, government and academic laboratories worldwide was performed as a benchmark using the NISTmAb, from the National Institute of Standards and Technology (NIST), to facilitate the translation of the 2D-NMR method into routine use for biopharmaceutical product development. Two-dimensional 1H,15N and 1H,13C NMR spectra were acquired with harmonized experimental protocols on the unlabeled Fab domain and a uniformly enriched-15N, 20%-13C-enriched system suitability sample derived from the NISTmAb. Chemometric analyses from over 400 spectral maps acquired on 39 different NMR spectrometers ranging from 500 MHz to 900 MHz demonstrate spectral fingerprints that are fit-for-purpose for the assessment of HOS. The 2D-NMR method is shown to provide the measurement reliability needed to move the technique from an emerging technology to a harmonized, routine measurement that can be generally applied with great confidence to high precision assessments of the HOS of mAb-based biotherapeutics.

19F NMR studies provide insights into lipid membrane interactions of listeriolysin O, a pore forming toxin from Listeria monocytogenes.

Listeria monocytogenes is a mammalian pathogen that causes gastroenteritis, miscarriages and infections of the central nervous system in immunocompromised individuals. Its main virulence factor is listeriolysin O (LLO), a pore-forming cholesterol-dependent cytolysin (CDC), which enables bacterial escape from the phagolysosome and contributes to bacterial pathogenicity. Details of cholesterol (Chol) recognition and membrane binding mechanisms by LLO are still not known. Here we used 19F-NMR spectroscopy in order to assess LLO-Chol interactions in solution and in a Chol-rich membrane environment. LLO has six tryptophan residues located in the region of the molecule that is first in contact with lipid membranes. 19F-LLO, which contained 5-fluoro-tryptophans, was prepared by using isotopic labelling in an E. coli expression system. Signals in the 19F-NMR spectrum of 19F-LLO were unambiguously assigned by using a series of single Trp → Phe point mutations. The results employing various cholesterol preparations in solution indicate that tryptophan residues are not directly involved in Chol binding in solution. However, significant chemical shift changes were observed upon LLO binding to Chol-rich membranes, highlighting the role of tryptophan residues in membrane interactions (W512) and oligomerisation (W189 and W489).

Analysis of Prion Protein Structure Using Nuclear Magnetic Resonance Spectroscopy.

Nuclear magnetic resonance (NMR) spectroscopy is a powerful experimental tool for obtaining information on three-dimensional (3D) structures of proteins at atomic resolution. In inherited forms of prion diseases, misfolding of cellular prion protein, PrPC, into its pathological form, PrPSc, is caused by mutations in the human prion protein gene (PRNP). Understanding of the earliest stages of the conformational changes leading to spontaneous generation of prions in inherited forms of prion diseases may benefit from detailed structural analysis of different human (Hu) PrP variants. Here, we describe the protocol for structure determination of HuPrP variants by NMR spectroscopy in solution that consists of preparation of NMR samples, acquisition of NMR data, NMR resonance assignments, and structure calculation.

Understanding the Effect of Disease-Related Mutations on Human Prion Protein Structure: Insights From NMR Spectroscopy.

Prion diseases or transmissible spongiform encephalopathies constitute a group of fatal neurodegenerative diseases that can be of sporadic, genetic, or acquired origin. The central molecular event of prion diseases is the conformational conversion of the physiological cellular prion protein, PrPC, into a disease-associated form known as prion or PrPSc. Spontaneous generation of prions in genetic prion diseases is caused by mutations in the human prion protein gene (PRNP). Understanding of the earliest conformational changes during misfolding of PrPC in genetic forms of prion diseases may benefit from detailed structural characterization of various human (Hu) PrP variants. Nuclear magnetic resonance (NMR) spectroscopy offers unique opportunities to obtain detailed atomic-level structure information. In this chapter we present an overview of high-resolution NMR studies on several HuPrPs with disease-associated mutations at mildly acidic and physiological pH conditions that provided valuable insights into possible key structural determinants underlying the formation of prions.

Truncated prion protein PrP226* - A structural view on its role in amyloid disease.

In the brain of patients with transmissible spongiform encephalopathies, besides PrPSc aggregates, deposition of truncated PrP molecules was described. Jansen et al. reported two clinical cases with deposition of C-terminally truncated PrP, one of them ending with Tyr226. We have previously described the discovery of monoclonal antibody V5B2 that selectively recognizes this version of the prion protein, which we called PrP226*. Using monoclonal antibody V5B2 we showed that accumulation of PrP226* is characteristic for most types of human and animal TSEs. Its distribution correlates to the distribution of PrPSc aggregates. To gain insight into the structural basis of its presence and distribution in PrP aggregates, we have determined the NMR structure of recombinant PrP226*. The structure of the protein consists of a disordered N-terminal part (residues 90-125) and a structured C-terminal part (residues 126-226). The C-terminal segment consists of four α-helices and a short antiparallel ß-sheet. Our model predicts a break in the C-terminal helix and reorganized hydrophobic interactions between helix α3 and ß2-α2 loop due to the shorter C-terminus. The structural model gives information on the possible role of the protein in the development of amyloid disease and can serve as a foundation to develop tools for prevention and treatment of prion diseases.

Biosimilar structural comparability assessment by NMR: from small proteins to monoclonal antibodies.

Biosimilar drug products must have a demonstrated similarity with respect to the reference product's molecules in order to ensure both the effectiveness of the drug and the patients' safety. In this paper the fusion framework of a highly sensitive NMR fingerprinting approach for conformational changes and mathematically-based biosimilarity metrics is introduced. The final goal is to translate the complex spectral information into biosimilarity scores, which are then used to estimate the degree of similarity between the biosimilar and the reference product. The proposed method was successfully applied to a small protein, i.e., filgrastim (neutropenia treatment), which is the first biosimilar approved in the United States, and a relatively large protein, i.e., monoclonal antibody rituximab (lymphoma treatment). This innovative approach introduces a new level of sensitivity to structural changes that are induced by, e.g., a small pH shift or other changes in the protein formulation.

The N Terminus of the Prion Protein Mediates Functional Interactions with the Neuronal Cell Adhesion Molecule (NCAM) Fibronectin Domain.

The cellular form of the prion protein (PrPC) is a highly conserved glycoprotein mostly expressed in the central and peripheral nervous systems by different cell types in mammals. A misfolded, pathogenic isoform, denoted as prion, is related to a class of neurodegenerative diseases known as transmissible spongiform encephalopathy. PrPC function has not been unequivocally clarified, and it is rather defined as a pleiotropic protein likely acting as a dynamic cell surface scaffolding protein for the assembly of different signaling modules. Among the variety of PrPC protein interactors, the neuronal cell adhesion molecule (NCAM) has been studied in vivo, but the structural basis of this functional interaction is still a matter of debate. Here we focused on the structural determinants responsible for human PrPC (HuPrP) and NCAM interaction using stimulated emission depletion (STED) nanoscopy, SPR, and NMR spectroscopy approaches. PrPC co-localizes with NCAM in mouse hippocampal neurons, and this interaction is mainly mediated by the intrinsically disordered PrPC N-terminal tail, which binds with high affinity to the NCAM fibronectin type-3 domain. NMR structural investigations revealed surface-interacting epitopes governing the interaction between HuPrP N terminus and the second module of the NCAM fibronectin type-3 domain. Our data provided molecular details about the interaction between HuPrP and the NCAM fibronectin domain, and revealed a new role of PrPC N terminus as a dynamic and functional element responsible for protein-protein interaction.

Structure and calcium-binding studies of calmodulin-like domain of human non-muscle α-actinin-1.

The activity of several cytosolic proteins critically depends on the concentration of calcium ions. One important intracellular calcium-sensing protein is α-actinin-1, the major actin crosslinking protein in focal adhesions and stress fibers. The actin crosslinking activity of α-actinin-1 has been proposed to be negatively regulated by calcium, but the underlying molecular mechanisms are poorly understood. To address this, we determined the first high-resolution NMR structure of its functional calmodulin-like domain (CaMD) in calcium-bound and calcium-free form. These structures reveal that in the absence of calcium, CaMD displays a conformationally flexible ensemble that undergoes a structural change upon calcium binding, leading to limited rotation of the N- and C-terminal lobes around the connecting linker and consequent stabilization of the calcium-loaded structure. Mutagenesis experiments, coupled with mass-spectrometry and isothermal calorimetry data designed to validate the calcium binding stoichiometry and binding site, showed that human non-muscle α-actinin-1 binds a single calcium ion within the N-terminal lobe. Finally, based on our structural data and analogy with other α-actinins, we provide a structural model of regulation of the actin crosslinking activity of α-actinin-1 where calcium induced structural stabilisation causes fastening of the juxtaposed actin binding domain, leading to impaired capacity to crosslink actin.

The cytosolic tail of the tumor marker protein Trop2--a structural switch triggered by phosphorylation.

Trop2 is a transmembrane signaling glycoprotein upregulated in stem and carcinoma cells. Proliferation-enhancing signaling involves regulated intramembrane proteolytic release of a short cytoplasmic fragment, which is later engaged in a cytosolic signaling complex. We propose that Trop2 function is modulated by phosphorylation of a specific serine residue within this cytosolic region (Ser303), and by proximity effects exerted on the cytosolic tail by Trop2 dimerization. Structural characterization of both the transmembrane (Trop2TM) and cytosolic regions (Trop2IC) support this hypothesis, and shows that the central region of Trop2IC forms an α-helix. Comparison of NMR structures of non-phosphorylated and phosphorylated forms suggest that phosphorylation of Trop2IC triggers salt bridge reshuffling, resulting in significant conformational changes including ordering of the C-terminal tail. In addition, we demonstrate that the cytosolic regions of two Trop2 subunits can be brought into close proximity via transmembrane part dimerization. Finally, we show that Ser303-phosphorylation significantly affects the structure and accessibility of functionally important regions of the cytosolic tail. These observed structural features of Trop2 at the membrane-cytosol interface could be important for regulation of Trop2 signaling activity.

NMR structural studies of human cellular prion proteins.

Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative disorders associated with the conformational conversion of the cellular prion protein, PrP(C), into a pathological form known as prion or PrP(Sc). They can be classified into sporadic, inherited and infectious forms. Spontaneous generation of PrP(Sc) in inherited forms of prion diseases is caused by mutations in the human prion protein gene (PRNP). A major goal in prion biology is unraveling the molecular mechanism by which PrP(C) misfolds and leads to development of diseases. Structural characterization of various human PrP (HuPrP) variants may be helpful for better understanding of the earliest stages of the conformational changes leading to spontaneous generation of prions. Here, we review the results of the recent high-resolution nuclear magnetic resonance (NMR) structural studies on HuPrPs with pathological Q212P and V210I mutations linked with Gerstmann-Sträussler-Scheinker (GSS) syndrome and familial Creutzfeldt-Jakob disease (fCJD), respectively, and HuPrP carrying naturally occurring E219K polymorphism considered to protect against sporadic CJD (sCJD). We describe subtle local differences between the three-dimensional (3D) structures of HuPrP mutants and the wild-type (WT) protein, providing new insights into the possible key structural determinants underlying conversion of PrP(C) into PrP(Sc). Also highlighted are the most recent findings from NMR studies about the effect of pH on the structural features of HuPrP with V210I mutation.

Probing early misfolding events in prion protein mutants by NMR spectroscopy.

The post-translational conversion of the ubiquitously expressed cellular form of the prion protein, PrPC, into its misfolded and pathogenic isoform, known as prion or PrPSc, plays a key role in prion diseases. These maladies are denoted transmissible spongiform encephalopathies (TSEs) and affect both humans and animals. A prerequisite for understanding TSEs is unraveling the molecular mechanism leading to the conversion process whereby most α-helical motifs are replaced by ß-sheet secondary structures. Importantly, most point mutations linked to inherited prion diseases are clustered in the C-terminal domain region of PrPC and cause spontaneous conversion to PrPSc. Structural studies with PrP variants promise new clues regarding the proposed conversion mechanism and may help identify "hot spots" in PrPC involved in the pathogenic conversion. These investigations may also shed light on the early structural rearrangements occurring in some PrPC epitopes thought to be involved in modulating prion susceptibility. Here we present a detailed overview of our solution-state NMR studies on human prion protein carrying different pathological point mutations and the implications that such findings may have for the future of prion research.

Structural rearrangements at physiological pH: nuclear magnetic resonance insights from the V210I human prion protein mutant.

A major focus in prion structural biology studies is unraveling the molecular mechanism leading to the structural conversion of PrP(C) to its pathological form, PrP(Sc). In our recent studies, we attempted to understand the early events of the conformational changes leading to PrP(Sc) using as investigative tools point mutations clustered in the open reading frame of the human PrP gene and linked to genetic forms of human prion diseases. In the work presented here, we investigate the effect of pH on the nuclear magnetic resonance (NMR) structure of recombinant human PrP (HuPrP) carrying the pathological V210I mutation responsible for familial Creutzfeldt-Jakob disease. The NMR structure of HuPrP(V210I) determined at pH 7.2 shows the same overall fold as the previously determined structure of HuPrP(V210I) at pH 5.5. It consists of a disordered N-terminal tail (residues 90-124) and a globular C-terminal domain (residues 125-231) comprising three α-helices and a short antiparallel ß-sheet. Detailed comparison of three-dimensional structures of HuPrP(V210I) at pH 7.2 and 5.5 revealed significant local structural differences, with the most prominent pH-related structural variations clustered in the α(2)-α(3) interhelical region, at the interface of the ß(1)-α(1) loop, in helices α(1) and α(3), and in the ß(2)-α(2) loop region. The detailed analysis of interactions among secondary structure elements suggests a higher degree of structural ordering of HuPrP(V210I) under neutral-pH conditions, thus implying that spontaneous misfolding of PrP(C) may occur under acidic-pH conditions in endosomal compartments.

Structural basis for the protective effect of the human prion protein carrying the dominant-negative E219K polymorphism.

The most common form of prion disease in humans is sCJD (sporadic Creutzfeldt-Jakob disease). The naturally occurring E219K polymorphism in the HuPrP (human prion protein) is considered to protect against sCJD. To gain insight into the structural basis of its protective influence we have determined the NMR structure of recombinant HuPrP (residues 90-231) carrying the E219K polymorphism. The structure of the HuPrP(E219K) protein consists of a disordered N-terminal tail (residues 90-124) and a well-structured C-terminal segment (residues 125-231) containing three α-helices and two short antiparallel ß-strands. Comparison of NMR structures of the wild-type and HuPrPs with pathological mutations under identical experimental conditions revealed that, although the global architecture of the protein remains intact, replacement of Glu²¹9 with a lysine residue introduces significant local structural changes. The structural findings of the present study suggest that the protective influence of the E219K polymorphism is due to the alteration of surface charge distribution, in addition to subtle structural rearrangements localized within the epitopes critical for prion conversion.

Toward the molecular basis of inherited prion diseases: NMR structure of the human prion protein with V210I mutation.

The development of transmissible spongiform encephalopathies (TSEs) is associated with the conversion of the cellular prion protein (PrP(C)) into a misfolded, pathogenic isoform (PrP(Sc)). Spontaneous generation of PrP(Sc) in inherited forms of disease is caused by mutations in gene coding for PrP (PRNP). In this work, we describe the NMR solution-state structure of the truncated recombinant human PrP (HuPrP) carrying the pathological V210I mutation linked to genetic Creutzfeldt-Jakob disease. The three-dimensional structure of V210I mutant consists of an unstructured N-terminal part (residues 90-124) and a well-defined C-terminal domain (residues 125-228). The C-terminal domain contains three α-helices (residues 144-156, 170-194 and 200-228) and a short antiparallel ß-sheet (residues 129-130 and 162-163). Comparison with the structure of the wild-type HuPrP revealed that although two structures share similar global architecture, mutation introduces some local structural differences. The observed variations are mostly clustered in the α(2)-α(3) inter-helical interface and in the ß(2)-α(2) loop region. Introduction of bulkier Ile at position 210 induces reorientations of several residues that are part of hydrophobic core, thus influencing α(2)-α(3) inter-helical interactions. Another important structural feature involves the alteration of conformation of the ß(2)-α(2) loop region and the subsequent exposure of hydrophobic cluster to solvent, which facilitates intermolecular interactions involved in spontaneous generation of PrP(Sc). The NMR structure of V210I mutant offers new clues about the earliest events of the pathogenic conversion process that could be used for the development of antiprion drugs.

NMR structure of the human prion protein with the pathological Q212P mutation reveals unique structural features.

Prion diseases are fatal neurodegenerative disorders caused by an aberrant accumulation of the misfolded cellular prion protein (PrP(C)) conformer, denoted as infectious scrapie isoform or PrP(Sc). In inherited human prion diseases, mutations in the open reading frame of the PrP gene (PRNP) are hypothesized to favor spontaneous generation of PrP(Sc) in specific brain regions leading to neuronal cell degeneration and death. Here, we describe the NMR solution structure of the truncated recombinant human PrP from residue 90 to 231 carrying the Q212P mutation, which is believed to cause Gerstmann-Sträussler-Scheinker (GSS) syndrome, a familial prion disease. The secondary structure of the Q212P mutant consists of a flexible disordered tail (residues 90-124) and a globular domain (residues 125-231). The substitution of a glutamine by a proline at the position 212 introduces novel structural differences in comparison to the known wild-type PrP structures. The most remarkable differences involve the C-terminal end of the protein and the beta(2)-alpha(2) loop region. This structure might provide new insights into the early events of conformational transition of PrP(C) into PrP(Sc). Indeed, the spontaneous formation of prions in familial cases might be due to the disruptions of the hydrophobic core consisting of beta(2)-alpha(2) loop and alpha(3) helix.

The structural effects of the Cys-S-S-Cys bridge exchange by the His-Cu(II)-His motif studied on natural peptides--a promising tool for natural compounds-based design.

A replacement of both Cys residues by His in oxytocin (OXT) sequence allows for the formation of the stable complex with the {NH(2), N(Im), N(Im(macrochelate))} binding mode at the physiological pH. The detailed potentiometric and spectroscopic studies on the Cu(II) complexes of [His(1,6)]OXT, together with high resolution NMR investigations on 3D structures of Cu(II) complexes with [His(1,6)]OXT and [His(1,6)]AVP analogues are presented and discussed. Exchange of the Cys-S-S-Cys bridge by the His-Cu(II)-His motif is very promising, because the resulting complexes retain topological similarity to the native S-S bridged AVP and OXT at pH values corresponding to the physiological pH.