Sparse isotope labeling for nuclear magnetic resonance (NMR) of glycoproteins using 13C-glucose.

Preparation of samples for nuclear magnetic resonance (NMR) characterization of larger proteins requires enrichment with less abundant, NMR-active, isotopes such as 13C and 15N. This is routine for proteins that can be expressed in bacterial culture where low-cost isotopically enriched metabolic substrates can be used. However, it can be expensive for glycosylated proteins expressed in mammalian culture where more costly isotopically enriched amino acids are usually used. We describe a simple, relatively inexpensive procedure in which standard commercial media is supplemented with 13C-enriched glucose to achieve labeling of all glycans plus all alanines of the N-terminal domain of the highly glycosylated protein, CEACAM1. We demonstrate an ability to detect partially occupied N-glycan sites, sites less susceptible to processing by an endoglycosidase, and some unexpected truncation of the amino acid sequence. The labeling of both the protein (through alanines) and the glycans in a single culture requiring no additional technical expertise past standard mammalian expression requirements is anticipated to have several applications, including structural and functional screening of the many glycosylated proteins important to human health.

Complete thermodynamic and kinetic characterization of the isomer-specific interaction between Pin1-WW domain and the amyloid precursor protein cytoplasmic tail phosphorylated at Thr668.

Peptidyl prolyl cis-trans isomerization acts as an effective molecular timer that plays significant roles in biological and pathological processes. Enzymes such as Pin1 catalyze cis-trans isomerization, accelerating the otherwise slow isomerization rate into time scales relevant for cellular signaling. Here we have combined NMR line shape analysis, fluorescence spectroscopy, and isothermal titration calorimetry to determine the kinetic and thermodynamic parameters describing the trans-specific interaction between the binding domain of Pin1 (WW domain) and a key cis-trans molecular switch in the amyloid precursor protein cytoplasmic tail. A three-state model, in which the cis-trans isomerization equilibrium is coupled to the binding equilibrium through the trans isomer, was found to fit the data well. The trans isomer binds the WW domain with ∼22 µM affinity via very fast association (approaching the diffusion limit) and dissociation rates. The common structural and electrostatic characteristics of Pin1 substrates, which contain a phosphorylated serine/threonine-proline motif, suggest that very rapid binding kinetics are a general feature of Pin1 interactions with other substrates. The fast binding kinetics of the WW domain allows rapid response of Pin1 to the dynamic events of phosphorylation and dephosphorylation in the cell that alter the relative populations of diverse Pin1 substrates. Furthermore, our results also highlight the vastly different rates at which slow uncatalyzed cis-trans isomerization and fast isomer-specific binding events occur. These results, along with the experimental methods presented herein, should guide future experiments aimed at the thermodynamic and kinetic characterization of cis-trans molecular switches and isomer-specific interactions involved in various biological processes.

Glycan Conformation in the Heavily Glycosylated Protein, CEACAM1.

Glycans attached to glycoproteins can contribute to stability, mediate interactions with other proteins, and initiate signal transduction. Glycan conformation, which is critical to these processes, is highly variable and often depicted as sampling a multitude of conformers. These conformers can be generated by molecular dynamics simulations, and more inclusively by accelerated molecular dynamics, as well as other extended sampling methods. However, experimental assessments of the contribution that various conformers make to a native ensemble are rare. Here, we use long-range pseudo-contact shifts (PCSs) of NMR resonances from an isotopically labeled glycoprotein to identify preferred conformations of its glycans. The N-terminal domain from human Carcinoembryonic Antigen Cell Adhesion Molecule 1, hCEACAM1-Ig1, was used as the model glycoprotein in this study. It has been engineered to include a lanthanide-ion-binding loop that generates PCSs, as well as a homogeneous set of three 13C-labeled N-glycans. Analysis of the PCSs indicates that preferred glycan conformers have extensive contacts with the protein surface. Factors leading to this preference appear to include interactions between N-acetyl methyls of GlcNAc residues and hydrophobic surface pockets on the protein surface.

AssignSLP_GUI, a software tool exploiting AI for NMR resonance assignment of sparsely labeled proteins.

Not all proteins are amenable to uniform isotopic labeling with 13C and 15N, something needed for the widely used, and largely deductive, triple resonance assignment process. Among them are proteins expressed in mammalian cell culture where native glycosylation can be maintained, and proper formation of disulfide bonds facilitated. Uniform labeling in mammalian cells is prohibitively expensive, but sparse labeling with one or a few isotopically enriched amino acid types is an option for these proteins. However, assignment then relies on accessing the best match between a variety of measured NMR parameters and predictions based on 3D structure, often from X-ray crystallography. Finding this match is a challenging process that has benefitted from many computational tools, including trained neural nets for chemical shift prediction, genetic algorithms for searches through a myriad of assignment possibilities, and now AI-based prediction of high-quality structures for protein targets. AssignSLP_GUI, a new version of a software package for assignment of resonances from sparsely-labeled proteins, uses many of these tools. These tools and new additions to the package are highlighted in an application to a sparsely-labeled domain from a glycoprotein, CEACAM1.

Validated determination of NRG1 Ig-like domain structure by mass spectrometry coupled with computational modeling.

High resolution hydroxyl radical protein footprinting (HR-HRPF) is a mass spectrometry-based method that measures the solvent exposure of multiple amino acids in a single experiment, offering constraints for experimentally informed computational modeling. HR-HRPF-based modeling has previously been used to accurately model the structure of proteins of known structure, but the technique has never been used to determine the structure of a protein of unknown structure. Here, we present the use of HR-HRPF-based modeling to determine the structure of the Ig-like domain of NRG1, a protein with no close homolog of known structure. Independent determination of the protein structure by both HR-HRPF-based modeling and heteronuclear NMR was carried out, with results compared only after both processes were complete. The HR-HRPF-based model was highly similar to the lowest energy NMR model, with a backbone RMSD of 1.6 Å. To our knowledge, this is the first use of HR-HRPF-based modeling to determine a previously uncharacterized protein structure.

Complete determination of the Pin1 catalytic domain thermodynamic cycle by NMR lineshape analysis.

The phosphorylation-specific peptidyl-prolyl isomerase Pin1 catalyzes the isomerization of the peptide bond preceding a proline residue between cis and trans isomers. To best understand the mechanisms of Pin1 regulation, rigorous enzymatic assays of isomerization are required. However, most measures of isomerase activity require significant constraints on substrate sequence and only yield rate constants for the cis isomer, [Formula: see text] and apparent Michaelis constants, [Formula: see text]. By contrast, NMR lineshape analysis is a powerful tool for determining microscopic rates and populations of each state in a complex binding scheme. The isolated catalytic domain of Pin1 was employed as a first step towards elucidating the reaction scheme of the full-length enzyme. A 24-residue phosphopeptide derived from the amyloid precurser protein intracellular domain (AICD) phosphorylated at Thr668 served as a biologically-relevant Pin1 substrate. Specific (13)C labeling at the Pin1-targeted proline residue provided multiple reporters sensitive to individual isomer binding and on-enzyme catalysis. We have performed titration experiments and employed lineshape analysis of phosphopeptide (13)C-(1)H constant time HSQC spectra to determine [Formula: see text], [Formula: see text], [Formula: see text], and [Formula: see text] for the catalytic domain of Pin1 acting on this AICD substrate. The on-enzyme equilibrium value of [E·trans]/[E·cis] = 3.9 suggests that the catalytic domain of Pin1 is optimized to operate on this substrate near equilibrium in the cellular context. This highlights the power of lineshape analysis for determining the microscopic parameters of enzyme catalysis, and demonstrates the feasibility of future studies of Pin1-PPIase mutants to gain insights on the catalytic mechanism of this important enzyme.

Neighboring phosphoSer-Pro motifs in the undefined domain of IRAK1 impart bivalent advantage for Pin1 binding.

The peptidyl prolyl isomerase Pin1 has two domains that are considered to be its binding (WW) and catalytic (PPIase) domains, both of which interact with phosphorylated Ser/Thr-Pro motifs. This shared specificity might influence substrate selection, as many known Pin1 substrates have multiple sequentially close phosphoSer/Thr-Pro motifs, including the protein interleukin-1 receptor-associated kinase-1 (IRAK1). The IRAK1 undefined domain (UD) contains two sets of such neighboring motifs (Ser131/Ser144 and Ser163/Ser173), suggesting possible bivalent interactions with Pin1. Using a series of NMR titrations with 15N-labeled full-length Pin1 (Pin1-FL), PPIase, or WW domain and phosphopeptides representing the Ser131/Ser144 and Ser163/Ser173 regions of IRAK1-UD, bivalent interactions were investigated. Binding studies using singly phosphorylated peptides showed that individual motifs displayed weak affinities (> 100 µm) for Pin1-FL and each isolated domain. Analysis of dually phosphorylated peptides binding to Pin1-FL showed that inclusion of bivalent states was necessary to fit the data. The resulting complex model and fitted parameters were applied to predict the impact of bivalent states at low micromolar concentrations, demonstrating significant affinity enhancement for both dually phosphorylated peptides (3.5 and 24 µm for peptides based on the Ser131/Ser144 and Ser163/Ser173 regions, respectively). The complementary technique biolayer interferometry confirmed the predicted affinity enhancement for a representative set of singly and dually phosphorylated Ser131/Ser144 peptides at low micromolar concentrations, validating model predictions. These studies provide novel insights regarding the complexity of interactions between Pin1 and activated IRAK1, and more broadly suggest that phosphorylation of neighboring Ser/Thr-Pro motifs in proteins might provide competitive advantage at cellular concentrations for engaging with Pin1.