ABSTRACT
Parkinson's Disease (PD) affects millions of people worldwide with no cure to halt the progress of the disease. Leucine-rich repeat kinase 2 (LRRK2) is the most common genetic cause of PD and, as such, LRRK2 inhibitors are promising therapeutic agents. In the last decade, great progress in the LRRK2 field has been made. This review provides a comprehensive overview of the current state of the art, presenting recent developments and challenges in developing LRRK2 inhibitors, and discussing extensively the potential targeting strategies from the protein perspective. As currently there are three LRRK2-targeting agents in clinical trials, more developments are predicted in the upcoming years.
Subject(s)
Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/metabolism , Parkinson Disease/drug therapy , Allosteric Site , Animals , Biomarkers/metabolism , GTP Phosphohydrolases/metabolism , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/antagonists & inhibitors , Mice , Monomeric GTP-Binding Proteins/metabolism , Neurodegenerative Diseases/metabolism , Parkinson Disease/metabolism , Phosphorylation , Protein Conformation , Protein Domains , Protein Interaction Mapping , Treatment OutcomeABSTRACT
Metallothioneins (MTs), small cysteine-rich proteins, present in four major isoforms, are key proteins involved in zinc and copper homeostasis in mammals. To date, only one X-ray crystal structure of a MT has been solved. It demonstrates seven bivalent metal ions bound in two structurally independent domains with M4S11 (α) and M3S9 (ß) clusters. Recent discoveries indicate that Zn(ii) ions are bound with MT2 with the range from nano- to picomolar affinity, which determines its cellular zinc buffering properties that are demonstrated by the presence of partially Zn(ii)-depleted MT2 species. These forms serve as Zn(ii) donors or acceptors and are formed under varying cellular free Zn(ii) concentrations. Due to the lack of appropriate methods, knowledge regarding the structure of partially-depleted metallothionein is lacking. Here, we describe the Zn(ii) binding mechanism in human MT2 with high resolution with respect to particular Zn(ii) binding sites, and provide structural insights into Zn(ii)-depleted MT species. The results were obtained by the labelling of metal-free cysteine residues with iodoacetamide and subsequent top-down electrospray ionization analysis, MALDI MS, bottom-up nanoLC-MALDI-MS/MS approaches and molecular dynamics (MD) simulations. The results show that the α-domain is formed sequentially in the first stages, followed by the formation of the ß-domain, although both processes overlap, which is in contrast to the widely investigated cadmium MT. Independent ZnS4 cores are characteristic for early stages of domain formation and are clustered in later stages. However, Zn-S network rearrangement in the ß-domain upon applying the seventh Zn(ii) ion explains its lower affinity. Detailed analysis showed that the weakest Zn(ii) ion associates with the ß-domain by coordination to Cys21, which was also found to dissociate first in the presence of the apo-form of sorbitol dehydrogenase. We found that Zn(ii) binding to the isolated ß-domain differs significantly from the whole protein, which explains its previously observed different Zn(ii)-binding properties. MD results obtained for Zn(ii) binding to the whole protein and isolated ß-domain are highly convergent with mass spectrometry data. This study provides a comprehensive overview of the crosstalk of structural and zinc buffering related-to-thermodynamics properties of partially metal-saturated mammalian MT2 and sheds more light on other MT proteins and zinc homeostasis.
Subject(s)
Coordination Complexes/metabolism , Metallothionein/metabolism , Zinc/metabolism , Alkylation , Coordination Complexes/chemistry , Humans , Metallothionein/chemistry , Models, Molecular , Molecular Dynamics Simulation , Protein Conformation , Zinc/chemistryABSTRACT
The binding of metal ions at the interface of protein complexes presents a unique and poorly understood mechanism of molecular assembly. A remarkable example is the Rad50 zinc hook domain, which is highly conserved and facilitates the Zn2+-mediated homodimerization of Rad50 proteins. Here, we present a detailed analysis of the structural and thermodynamic effects governing the formation and stability (logK12 = 20.74) of this evolutionarily conserved protein assembly. We have dissected the determinants of the stability contributed by the small ß-hairpin of the domain surrounding the zinc binding motif and the coiled-coiled regions using peptides of various lengths from 4 to 45 amino acid residues, alanine substitutions and peptide bond-to-ester perturbations. In the studied series of peptides, an >650 000-fold increase of the formation constant of the dimeric complex arises from favorable enthalpy because of the increased acidity of the cysteine thiols in metal-free form and the structural properties of the dimer. The dependence of the enthalpy on the domain fragment length is partially compensated by the entropic penalty of domain folding, indicating enthalpy-entropy compensation. This study facilitates understanding of the metal-mediated protein-protein interactions in which the metal ion is critical for the tight association of protein subunits.
Subject(s)
Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/metabolism , Exodeoxyribonucleases/chemistry , Exodeoxyribonucleases/metabolism , Pyrococcus furiosus/enzymology , Zinc/metabolism , Amino Acid Motifs , Amino Acid Substitution , Archaeal Proteins/genetics , Binding Sites , Crystallography, X-Ray , Endodeoxyribonucleases/genetics , Entropy , Exodeoxyribonucleases/genetics , Models, Molecular , Protein Binding , Protein Domains , Protein Folding , Protein Multimerization , Protein Stability , Pyrococcus furiosus/chemistry , Pyrococcus furiosus/geneticsABSTRACT
Two crystal structures of urethane-protected derivatives of aspartic acid dimethyl ester are presented, namely dimethyl (2S)-2-[(tert-butoxycarbonyl)amino]butanedioate, C(11)H(19)NO(6), and dimethyl (2S)-2-{bis[(tert-butoxycarbonyl]amino}butanedioate, C(16)H(27)NO(8). The geometry at the N atom is discussed and compared with similar structures. The analysis of singly and doubly N-substituted derivatives reveals an elongation of all bonds involving the N atom and conformational changes of the amino acid side chain due to steric interactions with two bulky substituents on the amino group.
ABSTRACT
Derivatization of peptides as quaternary ammonium salts (QAS) is a known method for sensitive detection by electrospray ionization tandem mass spectrometry. Hydrogens at α-carbon atom in N,N,N-trialkylglycine residue can be easily exchanged by deuterons. The exchange reaction is base-catalyzed and is dramatically slow at lower pH. Introduced deuterons are stable in acidic aqueous solution and are not back-exchanged during LC-MS analysis. Increased ionization efficiency, provided by the fixed positive charge on QAS group, as well as the deuterium labeling, enables the analysis of trace amounts of peptides.
