Design of a calcium-binding protein with desired structure in a cell adhesion molecule

Ca2+, "a signal of life and death", controls numerous cellular processes through interactions with proteins. An effective approach to understanding the role of Ca2+ is the design of a Ca2+-binding protein with predicted structural and functional properties. To design de novo Ca2+-binding sites in proteins is challenging due to the high coordination numbers and the incorporation of charged ligand residues, in addition to Ca2+-induced conformational change. Here, we demonstrate the successful design of a Ca2+-binding site in the non-Ca2+-binding cell adhesion protein CD2. This designed protein, Ca.CD2, exhibits selectivity for Ca2+ versus other di- and monovalent cations. In addition, La3+ (Kd 5.0 microM) and Tb3+ (Kd 6.6 microM) bind to the designed protein somewhat more tightly than does Ca2+ (Kd 1.4 mM). More interestingly, Ca.CD2 retains the native ability to associate with the natural target molecule. The solution structure reveals that Ca.CD2 binds Ca2+ at the intended site with the designed arrangement, which validates our general strategy for designing de novo Ca2+-binding proteins. The structural information also provides a close view of structural determinants that are necessary for a functional protein to accommodate the metal-binding site. This first success in designing Ca2+-binding proteins with desired structural and functional properties opens a new avenue in unveiling key determinants to Ca2+ binding, the mechanism of Ca2+ signaling, and Ca2+-dependent cell adhesion, while avoiding the complexities of the global conformational changes and cooperativity in natural Ca2+-binding proteins. It also represents a major achievement toward designing functional proteins controlled by Ca2+ binding.     

Artificial Peptide-Protein Necrosomes Promote Cell Death

The presence of disordered region or large interacting surface within proteins significantly challenges the development of targeted drugs, commonly known as the “undruggable” issue. Here, we report a heterogeneous peptide-protein assembling strategy to selectively phosphorylate proteins, thereby activating the necroptotic signaling pathway and promoting cell necroptosis. Inspired by the structures of natural necrosomes formed by receptor interacting protein kinases (RIPK) 1 and 3, the kinase-biomimetic peptides are rationally designed by incorporating natural or _D-amino acids, or connecting _D-amino acids in a retro-inverso (DRI) manner, leading to one RIPK3-biomimetic peptide PR3 and three RIPK1-biomimetic peptides. Individual peptides undergo self-assembly into nanofibrils, whereas mixing RIPK1-biomimetic peptides with PR3 accelerates and enhances assembly of PR3 . In particular, RIPK1-biomimetic peptide DRI-PR1 exhibits reliable binding affinity with protein RIPK3, resulting in specific cytotoxicity to colon cancer cells that overexpress RIPK3. Mechanistic studies reveal the increased phosphorylation of RIPK3 induced by RIPK1-biomimetic peptides, elucidating the activation of the necroptotic signaling pathway responsible for cell death without an obvious increase in secretion of inflammatory cytokines. Our findings highlight the potential of peptide-protein hybrid aggregation as a promising approach to address the “undruggable” issue and provide alternative strategies for overcoming cancer resistance in the future.     

Structural biology of the cell adhesion protein CD2: from molecular recognition to protein folding and design

CD2 (cluster of differentiation 2) is a cell adhesion molecule expressed on T cells and is recognized as a target for CD48 (rats) and CD58 (humans). Tremendous progress has been achieved in understanding the function of CD2, the mechanism of molecular recognition and protein folding, thus, leading towards the use of this protein as a scaffold for protein design. CD2 has been shown to set quantitative thresholds in T cell activation both in vivo and in vitro. Further, intracellular CD2 signaling pathways and networks are being discovered by the identification of several cytosolic tail binding proteins. In addition, a new method for directly measuring heterophilic adhesion has been developed. The functional "hot spot" for the adhesion surface of CD2 and CD58 has been dissected. Detailed NMR studies reveal that rat CD2 weakly self-associates to form a homodimeric structure in solution. Dynamic interaction of CD2 with the GYF and SH3 domains has been investigated. CD2 has been shown to form fibrils in the presence of 2,2,2-trifluoroethanol (TFE) and at low pH. Furthermore, kinetic studies have been completed to monitor the effect of surface hydrophobic residues and intramolecular bridges on the folding pathways of CD2. Our lab has de novo designed single calcium-binding sites into domain 1 of rat CD2 (CD2-D1) with strong metal selectivity. In addition, the EF-hand motifs have been grafted into CD2 to understand the site-specific calcium-binding affinity of calmodulin and calcium-dependent cell adhesion.     

Chick 28,000 Mr vitamin D-dependent calcium-binding protein in intestine, kidney and cerebellum. Purification using chromatofocusing

A purification method has been developed for chick 28,000 Mr vitamin D-dependent calcium-binding protein, involving Blue Sepharose CL-6B column chromatography, heat treatment and chromatofocusing with a microparticulate anion exchanger (Mono P). It allowed the rapid and reproducible purification of milligram amounts of homogeneous calcium-binding protein with good yields from chick intestine, kidney and cerebellum. The calcium-binding proteins thus obtained have the same molecular weight of 28,000, heat stability, calcium binding capability and apparent isoelectric point of 4.0. These physico-chemical properties are in good agreement with those of proteins isolated by a previous procedure, which gave a low and variable yield of calcium-binding protein.     

Design of a functional membrane protein by engineering a heme-binding site in glycophorin A

We have designed a functional model membrane protein by engineering a bis-Histidine heme-binding site into a natural membrane protein, glycophorin A (GpA), structurally characterized by the dimerization of a single transmembrane helix. Out of the 32 residues comprising the transmembrane helix of GpA, five amino acids were mutated; the resulting protein, ME1, has been characterized in dodecyl phosphocholin (DPC) micelles by UV-vis, CD spectroscopy, gel electrophoresis, and analytical ultracentrifugation. ME1 binds heme with sub-micromolar affinity and maintains the highly helical secondary structure and dimeric oligomerization state of GpA. The ME1-Heme complex exhibits a redox potential of -128 +/- 2 mV vs SHE, indicating that the heme resides in a hydrophobic environment and is well shielded from the aqueous phase. Moreover, ME1 catalyzes the hydrogen peroxide dependent oxidation of organic substrates such as TMB (2,2',5,5'-tetramethyl-benzidine). This protein may provide a useful framework to investigate how the protein matrix tunes the cofactor properties in membrane proteins.     

Effect of monovalent ion binding on molecular dynamics of the S100-family calcium-binding protein calbindin D9k

Calbindin D_9k is a member of the S100 subfamily of EF-hand calcium binding proteins, and has served as an important model system for biophysical studies. The fast timescale dynamics of the calcium-free (apo) state is characterized using molecular dynamics simulations. Order parameters for the backbone NH bond vectors are determined from the simulations and compared with experimentally derived values, with a focus on the dynamics of calcium-binding site I. There is a significant discrepancy between simulated and experimental order parameters for site I residues in the case of no ion bound in site I. However, it was found in the simulations that a Na⁺ ion can bind in site I, and the resulting order parameters determined from the simulations are in excellent agreement with experiment. Comparisons are made to X-ray structures of other S100 family members in which Na⁺ ions were observed or suggested to be bound in site I. © 2019 Wiley Periodicals, Inc.     

Engineering modular protein interaction switches by sequence overlap

Many cellular signaling pathways contain proteins whose interactions change in response to upstream inputs, allowing for conditional activation or repression of the interaction based on the presence of the input molecule. The ability to engineer similar regulation into protein interaction elements would provide us with powerful tools for controlling cell signaling. Here we describe an approach for engineering diverse synthetic protein interaction switches. Specifically, by overlapping the sequences of pairs of protein interaction domains and peptides, we have been able to generate mutually exclusive regulation over their interactions. Thus, the hybrid protein (which is composed of the two overlapped interaction modules) can bind to either of the two respective ligands for those modules, but not to both simultaneously. We show that these synthetic switch proteins can be used to regulate specific protein-protein interactions in vivo. These switches allow us to disrupt an interaction with the addition or activation of a protein input that has no natural connection to the interaction in question. Therefore, they give us the ability to make novel connections between normally unrelated signaling pathways and to rewire the input/output relationships of cellular behaviors. Our experiments also suggest a possible mechanism by which complex regulatory proteins might have evolved from simpler components.     

Protein-cation interactions: structural and thermodynamic aspects

Cations are specifically recognized by numerous proteins. Cations may play a structural role, as cofactors stabilizing their binding partners, or a functional role, as cofactors activating their binding partners or being themselves involved in enzymatic reactions. Despite their small size, their charge density and their specific interaction with highly charged residues allow them to induce significant conformational changes on their binding proteins. The protein conformational change induced by cation binding may be as large as to account for the complete folding of a protein (as evidenced in Hepatitis C NS3 protease, or human rhinovirus 2A protease), and they may also trigger oligomerization (as in calcium-binding protein 1). Especially intriguing is the ability of cation-binding proteins of discriminating between very similar cations. In particular, calcium and magnesium are recognized by proteins with markedly different binding affinities and cause significantly different conformational changes and stabilization effects in the binding proteins (as in the fifth ligand binding repeat of the LDL receptor binding domain, calcium-binding protein 1, or parvalbumin). This article summarizes recent findings on the structural and energetic impact of cation binding to different proteins. A general framework can be envisaged in which cations can be considered as a special type of allosteric effectors able to modulate the functional properties of proteins, in particular the ability to interact with biological targets, by altering their conformational equilibrium.     

Coupling caspase cleavage and proteasomal degradation of proteins carrying PEST motif

The degradation is critical to activation and deactivation of regulatory proteins involved in signaling pathways to cell growth, differentiation, stress responses and physiological cell death. Proteins carry domains and sequence motifs that function as prerequisite for their proteolysis by either individual proteases or the 26S multicomplex proteasomes. Two models for entry of substrates into the proteasomes have been considered. In one model, it is proposed that the ubiquitin chain attached to the protein serves as recognition element to drag them into the 19S regulatory particle, which promotes the unfolding required to its access into the 20S catalytic chamber. In second model, it is proposed that an unstructured tail located at amino or carboxyl terminus directly track proteins into the 26S/20S proteasomes. Caspases are cysteinyl aspartate proteases that control diverse signaling pathways, promoting the cleavage at one or two sites of hundreds of structural and regulatory protein substrates. Caspase cleavage sites are commonly found within PEST motifs, which are segments rich in proline (P), glutamic acid (D), aspartic acid (E) and serine (S) or threonine (T) residues. Considering that N- and C- terminal peptide carrying PEST motifs form disordered loops in the globular proteins after caspase cleavage, it is postulated here that these exposed termini serve as unstructured initiation site, coupling caspase cleavage and ubiquitin-proteasome dependent and independent degradation of short-lived proteins. This could explain the inherent susceptibility to proteolysis among proteins containing PEST motif.     

Protein-X,Pancreatic Stone-, Pancreatic thread-, reg-protein,P19, lithostathine,and now what? Characterization,structural analysis and putative function(s) of the major non-enzymatic protein of pancreatic secretions

Reg protein was first found in pancreatic stones. It was named Pancreatic Stone Protein and later renamed lithostathine, as it was assumed to prevent stone formation. The 144 amino acid protein is O-glycosylated on Thr-5. The glycan chain is variable in length and in charge. Lithostathine 3-D organization is of the C-lectin type, even though it is unlikely to have any functional calcium-binding site. The Arg11-Ile12 bond is readily cleaved by trypsin; the resulting C-terminal polypeptide precipitates at physiological pH and tends to form fibrils. The protein was more recently found in the regenerating endocrine pancreas and it was named Reg (for regenerating) protein. Numerous proteins related to Reg have been identified successively in several mammalian species. They constitute the Reg superfamily. Reg genes show the same organization and are located in the same chromosome region. These genes are therefore likely to derive from a common ancestor gene by duplication. In the course of evolution, they may have diverged in tissue-related expression and function. In the endocrine pancreas, Reg protein stimulates islet beta-cell growth and reduces experimental diabetes via the activation of a high affinity receptor. The role of the protein produced by the exocrine pancreas, however, is controversial. Not only is Reg/lithostathine unlikely to be a physiologically relevant pancreatic stone inhibitor, but it may contribute to stone formation. We suggest that it might help prevent the harmful activation of protease precursors in the pancreatic juice. The protein provides a useful model for examining the conformational changes associated with globular to fibril transformation.     

Potential of fragment recombination for rational design of proteins

It is hypothesized that protein domains evolved from smaller intrinsically stable subunits via combinatorial assembly. Illegitimate recombination of fragments that encode protein subunits could have quickly led to diversification of protein folds and their functionality. This evolutionary concept presents an attractive strategy to protein engineering, e.g., to create new scaffolds for enzyme design. We previously combined structurally similar parts from two ancient protein folds, the (βα)(8)-barrel and the flavodoxin-like fold. The resulting "hopeful monster" differed significantly from the intended (βα)(8)-barrel fold by an extra β-strand in the core. In this study, we ask what modifications are necessary to form the intended structure and what potential this approach has for the rational design of functional proteins. Guided by computational design, we optimized the interface between the fragments with five targeted mutations yielding a stable, monomeric protein whose predicted structure was verified experimentally. We further tested binding of a phosphorylated compound and detected that some affinity was already present due to an intact phosphate-binding site provided by one fragment. The affinity could be improved quickly to the level of natural proteins by introducing two additional mutations. The study illustrates the potential of recombining protein fragments with unique properties to design new and functional proteins, offering both a possible pathway of protein evolution and a protocol to rapidly engineer proteins for new applications.     

A comprehensive characterization of the T-cell antigen receptor complex composition by microcapillary liquid chromatography-tandem mass spectrometry

It has become apparent that many intracellular signaling processes involve the dynamic reorganization of cellular proteins into complex signaling assemblies that have a specific subunit composition, function, and subcellular location. Since the elements of such assemblies interact physically, multiprotein signaling complexes can be isolated and analyzed. Recent technical advances in highly sensitive protein identification by electrospray-tandem mass spectrometry have dramatically increased the sensitivity with which such analyses can be performed. The T-cell antigen receptor (TCR) is an oligomeric transmembrane protein complex that is essential to T-cell recognition and function. The extracellular protein domains are responsible for ligand binding while intracellular domains generate and transduce signals in response to specific receptor-ligand interactions. We used microbore capillary chromatography-tandem mass spectrometry to investigate the composition of the TCR protein complex isolated from resting and activated cells of the murine T-cell line CD11.3. We identified all the previously known subunits of the TCR/CD3 complex as well as proteins previously not known to associate with the TCR. The catalytic activities of some of these proteins could potentially be used to interfere pharmacologically with TCR signaling.     

Activation of protein phosphatase 1 by a small molecule designed to bind to the enzyme's regulatory site

The activity of protein phosphatase 1 (PP1), a serine-threonine phosphatase that participates ubiquitously in cellular signaling, is controlled by a wide variety of regulatory proteins that interact with PP1 at an allosteric regulatory site that recognizes a "loose" consensus sequence (usually designated as RVXF) found in all such regulatory proteins. Peptides containing the regulatory consensus sequence have been found to recapitulate the binding and PP1 activity modulation of the regulatory proteins, suggesting that it might be possible to design small-molecule surrogates that activate PP1 rather than inhibiting it. This prospect constitutes a largely unexplored way of controlling signaling pathways that could be functionally complementary to the much more extensively explored stratagem of kinase inhibition. Based on these principles, we have designed a microcystin analog that activates PP1.     

Rewiring kinase specificity with a synthetic adaptor protein

Signaling cascades are managed in time and space by interactions between and among proteins. These interactions are often aided by adaptor proteins, which guide enzyme-substrate pairs into proximity. Miniature proteins are a class of small, well-folded protein domains possessing engineered binding properties. Here we made use of two miniature proteins with complementary binding properties to create a synthetic adaptor protein that effectively redirects a ubiquitous signaling event: tyrosine phosphorylation. We report that miniature-protein-based adaptor 3 uses templated catalysis to redirect the Src family kinase Hck to phosphorylate hDM2, a negative regulator of the p53 tumor suppressor and a poor Hck substrate. Phosphorylation occurs with multiple turnover and at a single site targeted by c-Abl kinase in the cell.     

Designing Molecular Spanners to Throw in the Protein Networks

Proteins govern most aspects of cellular life and, through specific interfaces, are typically involved in intricate protein–protein interaction (PPI) networks and signaling pathways. Subtle up- or downregulation of key protein functions and PPIs results in disease; still, the preferred option to contrast the role of a protein in disease and healthy conditions alike remains its outright shutdown through orthosteric ligands that block its active site. Here, we explore subtler alternatives to modulate proteins and PPIs. Driven by a view of proteins as dynamic entities, we discuss ways to identify allosteric binding sites, which, when targeted by tailored ligands, can induce significant changes in the active site of a protein, and lead to agonistic or antagonistic effects. We also summarize the selective regulation of specific PPIs—either direct or allosteric—and show that effects can be stabilizing as well as destabilizing, depending on how the conformational equilibrium of a protein is shifted.     

Two-dimensional gel electrophoresis maps of the proteome and phosphoproteome of primitively cultured rat mesangial cells

Mesangial cells (MC) play an important role in maintaining the structure and function of the glomerulus. The proliferation of MC is a prominent feature of many kinds of glomerular disease. The first reference 2-DE maps of rat mesangial cells (RMC), stained with silver staining or Pro-Q Diamond dye, have been established here to describe the proteome and phosphoproteome of RMC, respectively. A total of 157 selected protein spots, corresponding to 118 unique proteins, have been identified by MALDI-TOF-MS or LC-ESI-IT-MS/MS, in which 37 protein spots representing 28 unique proteins have also been stained with Pro-Q Diamond, indicating that they are in phosphorylated forms. All the identified proteins were bioinformatically annotated in detail according to their physiochemical characteristics, subcellular location, and function. Most of the separated or identified protein spots are distributed in the area of mass 10-70 kDa and pI 5.0-8.0. The identified proteins include mainly cytoplasmic and nuclear proteins and some mitochondrial, endoplasmic reticulum, and membrane proteins. These proteins are classified into different functional groups such as structure and mobility proteins (21.2%), metabolic enzymes (16.9%), protein folding and metabolism proteins (13.6%), signaling proteins (14.4%), heat-shock proteins (7.6%), and other functional proteins (12.7%). While structure and mobility proteins are mostly represented by protein spots with high abundance, signaling proteins are mostly represented by protein spots with relatively low abundance. Such a 2-DE database for RMC, especially with many signaling proteins and phosphoproteins characterized, will provide a valuable resource for comparative proteomics analysis of normal and pathologic conditions affecting MC function or pathologic progress.     

Protein microparticles with controlled stability prepared via layer-by-layer adsorption of biopolyelectrolytes

This review covers the experimental data on the preparation and characterization of protein microparticles with controlled stability that are formed by layer-by-layer adsorption of oppositely charged macromolecules. Variants of using proteins as adsorbed polyelectrolyes, methods of incorporating proteins into matrixes (aggregates and microspheres) for further deposition of biopolyelectrolytes, and immobilization of proteins in preformed multilayered polyelectrolyte particles due to a change in the permeability of their shells are considered. Special attention is given to biocompatible and biodegradable microparticles characterized by depot functions, that is, the ability to reliably protect biologically active compounds from aggregative media of the body and to quantitatively release protein preparations (hormones, enzymes, and peptides) into solution when a certain acidity of solution is attained. This feature is especially important for designing peroral means of protein delivery.     

Affinity‐Labeling‐Based Introduction of a Reactive Handle for Natural Protein Modification

A new chemical method to site‐specifically modify natural proteins without the need for genetic manipulation is described. Our strategy involves the affinity‐labeling‐based attachment of a unique reactive handle at the surface of the target protein, and the subsequent selective transformation of the reactive handle by a bioorthogonal reaction to introduce a variety of functional probes into the protein. To demonstrate this approach, we synthesized labeling reagents that contain: 1) a benzenesulfonamide ligand that directs specifically to bovine carbonic anhydrase II (bCA), 2) an electrophilic epoxide group for protein labeling, 3) an exchangeable hydrazone bond linking the ligand and the epoxide group, and 4) an iodophenyl or acetylene handle. By incubating the labeling reagent with bCA, the reactive handle was covalently attached at the surface of bCA through epoxide ring opening. Either after or before removing the ligand by a hydrazone/oxime‐exhange reaction, which restores the enzymatic activity, the reactive handle incorporated could be derivatized by Suzuki coupling or Huisgen cycloaddition reactions. This method is also applicable to the target‐specific multiple modification in a protein mixture. The availability of various (photo)affinity‐labeling reagents and bioorthogonal reactions should extend the flexibility of this strategy for the site‐selective incorporation of many functional molecules into proteins.     

De novo design of a redox-active minimal rubredoxin mimic

Metal-binding sites in metalloproteins frequently occur at the interfaces of elements of secondary structure, which has enabled the retrostructural analysis of natural proteins and the de novo design of helical bundles that bind metal ion cofactors. However, the design of metalloproteins containing beta-structure is less well developed, despite the frequent occurrence of beta-conformations in natural metalloproteins. Here, we describe the design and construction of a beta-protein, RM1, that forms a stable, redox-active 4-Cys thiolate Fe(II/III) site analogous to the active site of rubredoxin. The protein folds into a beta-structure in the presence and absence of metal ions and binds Fe(II/III) to form a redox-active site that is stable to repeated cycles of oxidation and reduction, even in an aerobic environment.     

Regulating the Coordination State of a Heme Protein by a Designed Distal Hydrogen-Bonding Network

Heme coordination state determines the functional diversity of heme proteins. Using myoglobin as a model protein, we designed a distal hydrogen-bonding network by introducing both distal glutamic acid (Glu29) and histidine (His43) residues and regulated the heme into a bis-His coordination state with native ligands His64 and His93. This resembles the heme site in natural bis-His coordinated heme proteins such as cytoglobin and neuroglobin. A single mutation of L29E or F43H was found to form a distinct hydrogen-bonding network involving distal water molecules, instead of the bis-His heme coordination, which highlights the importance of the combination of multiple hydrogen-bonding interactions to regulate the heme coordination state. Kinetic studies further revealed that direct coordination of distal His64 to the heme iron negatively regulates fluoride binding and hydrogen peroxide activation by competing with the exogenous ligands. The new approach developed in this study can be generally applicable for fine-tuning the structure and function of heme proteins.