Alexander Goncearenco

A Dominant Negative Heterozygous G87R Mutation in the Zinc Transporter, ZnT-2 (SLC30A2), Results in Transient Neonatal Zinc Deficiency

Background: Infants of mothers carrying the H54R mutation in ZnT-2 develop transient neonatal zinc deficiency (TNZD). Results: Transfection of a novel heterozygous G87R mutant ZnT-2 resulted in its mislocalization, impaired zinc transport, and negative dominance. Conclusion: G87R is an inactivating mutation inflicting a dominant negative effect via homodimer formation. Significance: This study significantly advances our understanding regarding the molecular mechanism underlying TNZD. Zinc is an essential mineral, and infants are particularly vulnerable to zinc deficiency as they require large amounts of zinc for their normal growth and development. We have recently described the first loss-of-function mutation (H54R) in the zinc transporter ZnT-2 (SLC30A2) in mothers with infants harboring transient neonatal zinc deficiency (TNZD). Here we identified and characterized a novel heterozygous G87R ZnT-2 mutation in two unrelated Ashkenazi Jewish mothers with infants displaying TNZD. Transient transfection of G87R ZnT-2 resulted in endoplasmic reticulum-Golgi retention, whereas the WT transporter properly localized to intracellular secretory vesicles in HC11 and MCF-7 cells. Consequently, G87R ZnT-2 showed decreased stability compared with WT ZnT-2 as revealed by Western blot analysis. Three-dimensional homology modeling based on the crystal structure of YiiP, a close zinc transporter homologue from Escherichia coli, revealed that the basic arginine residue of the mutant G87R points toward the membrane lipid core, suggesting misfolding and possible loss-of-function. Indeed, functional assays including vesicular zinc accumulation, zinc secretion, and cytoplasmic zinc pool assessment revealed markedly impaired zinc transport in G87R ZnT-2 transfectants. Moreover, co-transfection experiments with both mutant and WT transporters revealed a dominant negative effect of G87R ZnT-2 over the WT ZnT-2; this was associated with mislocalization, decreased stability, and loss of zinc transport activity of the WT ZnT-2 due to homodimerization observed upon immunoprecipitation experiments. These findings establish that inactivating ZnT-2 mutations are an underlying basis of TNZD and provide the first evidence for the dominant inheritance of heterozygous ZnT-2 mutations via negative dominance due to homodimer formation.

SPACER: server for predicting allosteric communication and effects of regulation

The SPACER server provides an interactive framework for exploring allosteric communication in proteins with different sizes, degrees of oligomerization and function. SPACER uses recently developed theoretical concepts based on the thermodynamic view of allostery. It proposes easily tractable and meaningful measures that allow users to analyze the effect of ligand binding on the intrinsic protein dynamics. The server shows potential allosteric sites and allows users to explore communication between the regulatory and functional sites. It is possible to explore, for instance, potential effector binding sites in a given structure as targets for allosteric drugs. As input, the server only requires a single structure. The server is freely available at http://allostery.bii.a-star.edu.sg/.

Thermophilic Adaptation of Protein Complexes Inferred from Proteomic Homology Modeling

As protein complexes must remain in their native conformations at habitat temperatures, thermal adaptation requires adjustment of their parts and interactions between them. Based on independent sets of structural templates and sequences of 127 complete prokaryotic proteomes with optimal growth temperatures from 8 degrees C to 100 degrees C, we performed proteomic homology modeling of complexes and analyzed peculiarities in their traits related to thermal adaptation. We explore compositional determinants of thermostability of protein complexes based on the model of stability including negative and positive components of design. We show that positively charged amino acids play an important role in protein complexes, working in negative design against misfolded conformations and aberrant assemblies and contributing to positive design by stabilizing both the native interface and the overall structure of the complex. Aggregation propensity of interfaces is higher than that of surfaces and the difference between them increases with optimal growth temperature securing native complexes in hot environments.

Prototypes of elementary functional loops unravel evolutionary connections between protein functions

Motivation: Earlier studies of protein structure revealed closed loops with a characteristic size 25–30 residues and ring-like shape as a basic universal structural element of globular proteins. Elementary functional loops (EFLs) have specific signatures and provide functional residues important for binding/activation and principal chemical transformation steps of the enzymatic reaction. The goal of this work is to show how these functional loops evolved from pre-domain peptides and to find a set of prototypes from which the EFLs of contemporary proteins originated. Results: This article describes a computational method for deriving prototypes of EFLs based on the sequences of complete genomes. The procedure comprises the iterative derivation of sequence profiles followed by their hierarchical clustering. The scoring function takes into account information content on profile positions, thus preserving the signature. The statistical significance of scores is evaluated from the empirical distribution of scores of the background model. A set of prototypes of EFLs from archaeal proteomes is derived. This set delineates evolutionary connections between major functions and illuminates how folds and functions emerged in pre-domain evolution as a combination of prototypes. Contact: Igor.Berezovsky@uni.no

Protein function from its emergence to diversity in contemporary proteins.

The goal of this work is to learn from nature the rules that govern evolution and the design of protein function. The fundamental laws of physics lie in the foundation of the protein structure and all stages of the protein evolution, determining optimal sizes and shapes at different levels of structural hierarchy. We looked back into the very onset of the protein evolution with a goal to find elementary functions (EFs) that came from the prebiotic world and served as building blocks of the first enzymes. We defined the basic structural and functional units of biochemical reactions-elementary functional loops. The diversity of contemporary enzymes can be described via combinations of a limited number of elementary chemical reactions, many of which are performed by the descendants of primitive prebiotic peptides/proteins. By analyzing protein sequences we were able to identify EFs shared by seemingly unrelated protein superfamilies and folds and to unravel evolutionary relations between them. Binding and metabolic processing of the metal- and nucleotide-containing cofactors and ligands are among the most abundant ancient EFs that became indispensable in many natural enzymes. Highly designable folds provide structural scaffolds for many different biochemical reactions. We show that contemporary proteins are built from a limited number of EFs, making their analysis instrumental for establishing the rules for protein design. Evolutionary studies help us to accumulate the library of essential EFs and to establish intricate relations between different folds and functional superfamilies. Generalized sequence-structure descriptors of the EF will become useful in future design and engineering of desired enzymatic functions.

Exploring background mutational processes to decipher cancer genetic heterogeneity

Abstract Much remains unknown about the progression and heterogeneity of mutational processes in different cancers and their diagnostic and clinical potential. A growing body of evidence supports mutation rate dependence on the local DNA sequence context for various types of mutations. We propose several tools for the analysis of cancer context-dependent mutations, which are implemented in an online computational framework MutaGene. The framework explores DNA context-dependent mutational patterns and underlying somatic cancer mutagenesis, analyzes mutational profiles of cancer samples, identifies the combinations of underlying mutagenic processes including those related to infidelity of DNA replication and repair machinery, and various other endogenous and exogenous mutagenic factors. As a result, the combination of mutagenic processes can be identified in any query sample with subsequent comparison to mutational profiles derived from malignant and benign samples. In addition, mutagen or cancer-specific mutational background models are applied to calculate expected DNA and protein site mutability to decouple relative contributions of mutagenesis and selection in carcinogenesis, thus elucidating the site-specific driving events in cancer. MutaGene is freely available at https://www.ncbi.nlm.nih.gov/projects/mutagene/.

Nucleotide binding database NBDB – a collection of sequence motifs with specific protein-ligand interactions

NBDB database describes protein motifs, elementary functional loops (EFLs) that are involved in binding of nucleotide-containing ligands and other biologically relevant cofactors/coenzymes, including ATP, AMP, ATP, GMP, GDP, GTP, CTP, PAP, PPS, FMN, FAD(H), NAD(H), NADP, cAMP, cGMP, c-di-AMP and c-di-GMP, ThPP, THD, F-420, ACO, CoA, PLP and SAM. The database is freely available online at http://nbdb.bii.a-star.edu.sg. In total, NBDB contains data on 249 motifs that work in interactions with 24 ligands. Sequence profiles of EFL motifs were derived de novo from nonredundant Uniprot proteome sequences. Conserved amino acid residues in the profiles interact specifically with distinct chemical parts of nucleotide-containing ligands, such as nitrogenous bases, phosphate groups, ribose, nicotinamide, and flavin moieties. Each EFL profile in the database is characterized by a pattern of corresponding ligand–protein interactions found in crystallized ligand–protein complexes. NBDB database helps to explore the determinants of nucleotide and cofactor binding in different protein folds and families. NBDB can also detect fragments that match to profiles of particular EFLs in the protein sequence provided by user. Comprehensive information on sequence, structures, and interactions of EFLs with ligands provides a foundation for experimental and computational efforts on design of required protein functions.

Molecular mechanisms of adaptation emerging from the physics and evolution of nucleic acids and proteins

DNA, RNA and proteins are major biological macromolecules that coevolve and adapt to environments as components of one highly interconnected system. We explore here sequence/structure determinants of mechanisms of adaptation of these molecules, links between them, and results of their mutual evolution. We complemented statistical analysis of genomic and proteomic sequences with folding simulations of RNA molecules, unraveling causal relations between compositional and sequence biases reflecting molecular adaptation on DNA, RNA and protein levels. We found many compositional peculiarities related to environmental adaptation and the life style. Specifically, thermal adaptation of protein-coding sequences in Archaea is characterized by a stronger codon bias than in Bacteria. Guanine and cytosine load in the third codon position is important for supporting the aerobic life style, and it is highly pronounced in Bacteria. The third codon position also provides a tradeoff between arginine and lysine, which are favorable for thermal adaptation and aerobicity, respectively. Dinucleotide composition provides stability of nucleic acids via strong base-stacking in ApG dinucleotides. In relation to coevolution of nucleic acids and proteins, thermostability-related demands on the amino acid composition affect the nucleotide content in the second codon position in Archaea.

The fundamental tradeoff in genomes and proteomes of prokaryotes established by the genetic code, codon entropy, and physics of nucleic acids and proteins.

BackgroundMutations in nucleotide sequences provide a foundation for genetic variability, and selection is the driving force of the evolution and molecular adaptation. Despite considerable progress in the understanding of selective forces and their compositional determinants, the very nature of underlying mutational biases remains unclear.ResultsWe explore here a fundamental tradeoff, which analytically describes mutual adjustment of the nucleotide and amino acid compositions and its possible effect on the mutational biases. The tradeoff is determined by the interplay between the genetic code, optimization of the codon entropy, and demands on the structure and stability of nucleic acids and proteins.ConclusionThe tradeoff is the unifying property of all prokaryotes regardless of the differences in their phylogenies, life styles, and extreme environments. It underlies mutational biases characteristic for genomes with different nucleotide and amino acid compositions, providing foundation for evolution and adaptation.ReviewersThis article was reviewed by Eugene Koonin, Michael Gromiha, and Alexander Schleiffer.

Exploring the evolution of protein function in Archaea

BackgroundDespite recent progress in studies of the evolution of protein function, the questions what were the first functional protein domains and what were their basic building blocks remain unresolved. Previously, we introduced the concept of elementary functional loops (EFLs), which are the functional units of enzymes that provide elementary reactions in biochemical transformations. They are presumably descendants of primordial catalytic peptides.ResultsWe analyzed distant evolutionary connections between protein functions in Archaea based on the EFLs comprising them. We show examples of the involvement of EFLs in new functional domains, as well as reutilization of EFLs and functional domains in building multidomain structures and protein complexes.ConclusionsOur analysis of the archaeal superkingdom yields the dominating mechanisms in different periods of protein evolution, which resulted in several levels of the organization of biochemical function. First, functional domains emerged as combinations of prebiotic peptides with the very basic functions, such as nucleotide/phosphate and metal cofactor binding. Second, domain recombination brought to the evolutionary scene the multidomain proteins and complexes. Later, reutilization and de novo design of functional domains and elementary functional loops complemented evolution of protein function.