Alexei S. Kazakov

The impact of alpha-N-acetylation on structural and functional status of parvalbumin

The effect of alpha-N-acetylation (Nt-acetylation) on the properties of parvalbumin (PA), a Ca2+-binding relaxing factor of skeletal muscles and major food allergen, has been explored. Intact PA contains an N-terminal acetyl group which is absent in the protein expressed in Escherichia coli (rWT), as confirmed by mass spectrometry. Compared to intact pike α-PA, its rWT form exhibits essentially altered profile of thermal unfolding, lowered α-helicity, and decreased affinities to Ca2+ and Mg2+. The structural destabilization of the rWT protein results in lowered resistance to chymotryptic digestion and increased propensity to oligomerization. The rate constants of Ca2+ dissociation from the rWT PA are markedly increased, which indicates that Nt-acetylation modifies functional status of the protein. Rat α-PA demonstrates similar properties for intact and rWT forms. The drastic difference in the effects induced by Nt-acetylation in the PA orthologs can be rationalized by higher disorder level of AB domain in pike PA. Though evolution of PAs genes resulted in the protein sequences with highly divergent properties, Nt-acetylation unifies their functional properties. The structural stability conferred to PA by Nt-acetylation may contribute to its allergenicity. Overall, Nt-acetylation is shown to be a prerequisite for maintenance of structural and functional status of some parvalbumins.

Interleukin-11 binds specific EF-hand proteins via their conserved structural motifs

Interleukin-11 (IL-11) is a hematopoietic cytokine engaged in numerous biological processes and validated as a target for treatment of various cancers. IL-11 contains intrinsically disordered regions that might recognize multiple targets. Recently we found that aside from IL-11RA and gp130 receptors, IL-11 interacts with calcium sensor protein S100P. Strict calcium dependence of this interaction suggests a possibility of IL-11 interaction with other calcium sensor proteins. Here we probed specificity of IL-11 to calcium-binding proteins of various types: calcium sensors of the EF-hand family (calmodulin, S100B and neuronal calcium sensors: recoverin, NCS-1, GCAP-1, GCAP-2), calcium buffers of the EF-hand family (S100G, oncomodulin), and a non-EF-hand calcium buffer (α-lactalbumin). A specific subset of the calcium sensor proteins (calmodulin, S100B, NCS-1, GCAP-1/2) exhibits metal-dependent binding of IL-11 with dissociation constants of 1–19 μM. These proteins share several amino acid residues belonging to conservative structural motifs of the EF-hand proteins, ‘black’ and ‘gray’ clusters. Replacements of the respective S100P residues by alanine drastically decrease its affinity to IL-11, suggesting their involvement into the association process. Secondary structure and accessibility of the hinge region of the EF-hand proteins studied are predicted to control specificity and selectivity of their binding to IL-11. The IL-11 interaction with the EF-hand proteins is expected to occur under numerous pathological conditions, accompanied by disintegration of plasma membrane and efflux of cellular components into the extracellular milieu.

Structural Characterization of More Potent Alternatives to HAMLET, a Tumoricidal Complex of α-Lactalbumin and Oleic Acid

HAMLET is a complex of human α-lactalbumin (hLA) with oleic acid (OA) that kills various tumor cells and strains of Streptococcus pneumoniae. More potent protein-OA complexes were previously reported for bovine α-lactalbumin (bLA) and β-lactoglobulin (bLG), and pike parvalbumin (pPA), and here we explore their structural features. The concentration dependencies of the tryptophan fluorescence of hLA, bLA, and bLG complexes with OA reveal their disintegration at protein concentrations below the micromolar level. Chemical cross-linking experiments provide evidence that association with OA shifts the distribution of oligomeric forms of hLA, bLA, bLG, and pPA toward higher-order oligomers. This effect is confirmed for bLA and bLG using the dynamic light scattering method, while pPA is shown to associate with OA vesicles. Like hLA binding, OA binding increases the affinity of bLG for small unilamellar dipalmitoylphosphatidylcholine vesicles, while pPA efficiently binds to the vesicles irrespective of OA binding. The association of OA with bLG and pPA increases their α-helix and cross-β-sheet content and resistance to enzymatic proteolysis, which is indicative of OA-induced protein structuring. The lack of excess heat sorption during melting of bLG and pPA in complex with OA and the presence of a cooperative thermal transition at the level of their secondary structure suggest that the OA-bound forms of bLG and pPA lack a fixed tertiary structure but exhibit a continuous thermal transition. Overall, despite marked differences, the HAMLET-like complexes that were studied exhibit a common feature: a tendency toward protein oligomerization. Because OA-induced oligomerization has been reported for other proteins, this phenomenon is inherent to many proteins.

In search for globally disordered apo-parvalbumins: Case of parvalbumin β-1 from coho salmon

Parvalbumin (PA) is a classical EF-hand calcium-binding protein of muscle, neuronal, and other tissues, and a major fish allergen. Although certain apo-PAs lack tertiary structure, functional implications of that feature and its structural prerequisites remain unclear. In a search for unstable PAs, we probed conformational stability of parvalbumin β-1 from coho salmon (csPA), a cold water fish species, using circular dichroism, scanning calorimetry, hydrophobic probe fluorescence, limited proteolysis, chemical crosslinking and dynamic light scattering techniques. Apo-csPA is shown to be mainly monomeric protein with markedly disorganized secondary structure and lack of rigid tertiary structure. Examination of per-residue propensity for intrinsic disorder in the PA groups with either folded or unfolded apo-form using the average PONDR® VSL2P profiles revealed that the N-terminal region that includes α-helix A, AB-loop and N-terminal half of α-helix B is predicted to be less ordered in PAs with disordered apo-state. Application of the structural criteria developed for discrimination of disordered PAs indicate that the latter comprise about 16-19% of all PAs. We show that structural instability of apo-β-PA serves as a hallmark of elevated calcium affinity of the protein. Therefore, the successful predictions of unstable apo-PAs might facilitate search for PAs with maximal calcium affinity and possibly serving as calcium sensors.

Extremophilic 50S Ribosomal RNA-Binding Protein L35Ae as a Basis for Engineering of an Alternative Protein Scaffold

Due to their remarkably high structural stability, proteins from extremophiles are particularly useful in numerous biological applications. Their utility as alternative protein scaffolds could be especially valuable in small antibody mimetic engineering. These artificial binding proteins occupy a specific niche between antibodies and low molecular weight substances, paving the way for development of innovative approaches in therapeutics, diagnostics, and reagent use. Here, the 50S ribosomal RNA-binding protein L35Ae from the extremophilic archaea Pyrococcus horikoshii has been probed for its potential to serve as a backbone in alternative scaffold engineering. The recombinant wild type L35Ae has a native-like secondary structure, extreme thermal stability (mid-transition temperature of 90°C) and a moderate resistance to the denaturation by guanidine hydrochloride (half-transition at 2.6 M). Chemical crosslinking and dynamic light scattering data revealed that the wild type L35Ae protein has a propensity for multimerization and aggregation correlating with its non-specific binding to a model cell surface of HEK293 cells, as evidenced by flow cytometry. To suppress these negative features, a 10-amino acid mutant (called L35Ae 10X) was designed, which lacks the interaction with HEK293 cells, is less susceptible to aggregation, and maintains native-like secondary structure and thermal stability. However, L35Ae 10X also shows lowered resistance to guanidine hydrochloride (half-transition at 2.0M) and is more prone to oligomerization. This investigation of an extremophile protein’s scaffolding potential demonstrates that lowered resistance to charged chemical denaturants and increased propensity to multimerization may limit the utility of extremophile proteins as alternative scaffolds.

Supramolecular organization of Hfq-like proteins.

Bacterial Hfq proteins are structural homologs of archaeal and eukaryotic Sm/Lsm proteins, which are characterized by a 5-stranded β-sheet and an N-terminal α-helix. Previously, it was shown that archaeal Lsm proteins (SmAP) could produce long fibrils spontaneously, in contrast to the Hfq from Escherichia coli that could form similar fibrils only after special treatment. The organization of these fibrils is significantly different, but the reason for the dissimilarity has not been found. In the present work, we studied the process of fibril formation by bacterial protein Hfq from Pseudomonas aeruginosa and archaeal protein SmAP from Methanococcus jannaschii. Both proteins have high homology with E. coli Hfq. We found that Hfq from P. aeruginosa could form fibrils after substitutions in the conserved Sm2 motif only. SmAP from M. jannaschii, like other archaeal Lsm proteins, form fibrils spontaneously. Despite differences in the fibril formation conditions, the architecture of both was similar to that described for E. coli Hfq. Therefore, universal nature of fibril architecture formed by Hfq proteins is suggested.

Calcium-dependent interaction of monomeric S100P protein with serum albumin

S100 proteins are multifunctional (intra/extra)cellular mostly dimeric calcium-binding proteins engaged into numerous diseases. We have found that monomeric recombinant human S100P protein interacts with intact human serum albumin (HSA) in excess of calcium ions with equilibrium dissociation constant of 25-50nM, as evidenced by surface plasmon resonance spectroscopy and fluorescent titration by HSA of S100P labelled by fluorescein isothiocyanate. Calcium removal or S100P dimerization abolish the S100P-HSA interaction. The interaction is selective, since S100P does not bind bovine serum albumin and monomeric human S100B lacks interaction with HSA. In vitro glycation of HSA disables its binding to S100P. The revealed selective and highly specific conformation-dependent interaction between S100P and HSA shows that functional properties of monomeric and dimeric forms of S100 proteins are different, and raises concerns on validity of cell-based assays and animal models used for studies of (patho)physiological roles of extracellular S100 proteins.

Derivative of Extremophilic 50S Ribosomal Protein L35Ae as an Alternative Protein Scaffold

Small antibody mimetics, or alternative binding proteins (ABPs), extend and complement antibody functionality with numerous applications in research, diagnostics and therapeutics. Given the superiority of ABPs, the last two decades have witnessed development of dozens of alternative protein scaffolds (APSs) for the design of ABPs. Proteins from extremophiles with their high structural stability are especially favorable for APS design. Here, a 10X mutant of the 50S ribosomal protein L35Ae from hyperthermophilic archaea Pyrococcus horikoshii has been probed as an APS. A phage display library of L35Ae 10X was generated by randomization of its three CDR-like loop regions (repertoire size of 2×108). Two L35Ae 10X variants specific to a model target, the hen egg-white lysozyme (HEL), were isolated from the resulting library using phage display. The affinity of these variants (L4 and L7) to HEL ranges from 0.10 μM to 1.6 μM, according to surface plasmon resonance data. While L4 has 1–2 orders of magnitude lower affinity to HEL homologue, bovine α-lactalbumin (BLA), L7 is equally specific to HEL and BLA. The reference L35Ae 10X is non-specific to both HEL and BLA. L4 and L7 are more resistant to denaturation by guanidine hydrochloride compared to the reference L35Ae 10X (mid-transition concentration is higher by 0.1–0.5 M). Chemical crosslinking experiments reveal an increased propensity of L4 and L7 to multimerization. Overall, the CDR-like loop regions of L35Ae 10X represent a proper interface for generation of functional ABPs. Hence, L35Ae is shown to extend the growing family of protein scaffolds dedicated to the design of novel binding proteins.

Expression, Purification, and Characterization of Interleukin-11 Orthologues

Interleukin-11 (IL-11) is a multifunctional cytokine implicated in several normal and pathological processes. The decoding of IL-11 function and development of IL-11-targeted drugs dictate the use of laboratory animals and need of the better understanding of species specificity of IL-11 signaling. Here, we present a method for the recombinant interleukin-11 (rIL-11) production from the important model animals, mouse and macaque. The purified mouse and macaque rIL-11 interact with extracellular domain of human IL-11 receptor subunit α and activate STAT3 signaling in HEK293 cells co-expressing human IL-11 receptors with efficacies resembling those of human rIL-11. Hence, the evolutionary divergence does not impair IL-11 signaling. Furthermore, compared to human rIL-11 its macaque orthologue is 8-fold more effective STAT3 activator, which favors its use for treatment of thrombocytopenia as a potent substitute for human rIL-11. Compared to IL-6, IL-11 signaling exhibits lower species specificity, likely due to less conserved intrinsic disorder propensity within IL-6 orthologues. The developed express method for preparation of functionally active macaque/mouse rIL-11 samples is suited for exploration of the molecular mechanisms underlying IL-11 action and for development of the drug candidates for therapy of oncologic/hematologic/inflammatory diseases related to IL-11 signaling.