Galvanization of Protein-Protein Interactions in a Dynamic Zinc Interactome.

The presence of Zn2+ at protein-protein interfaces modulates complex function, stability, and introduces structural flexibility/complexity, chemical selectivity, and reversibility driven in a Zn2+-dependent manner. Recent studies have demonstrated that dynamically changing Zn2+ affects numerous cellular processes, including protein-protein communication and protein complex assembly. How Zn2+-involved protein-protein interactions (ZPPIs) are formed and dissociate and how their stability and reactivity are driven in a zinc interactome remain poorly understood, mostly due to experimental obstacles. Here, we review recent research advances on the role of Zn2+ in the formation of interprotein sites, their architecture, function, and stability. Moreover, we underline the importance of zinc networks in intersystemic communication and highlight bioinformatic and experimental challenges required for the identification and investigation of ZPPIs.

Drug Conjugation via Maleimide-Thiol Chemistry Does Not Affect Targeting Properties of Cysteine-Containing Anti-FGFR1 Peptibodies.

With a wide range of available cytotoxic therapeutics, the main focus of current cancer research is to deliver them specifically to the cancer cells, minimizing toxicity against healthy tissues. Targeted therapy utilizes different carriers for cytotoxic drugs, combining a targeting molecule, typically an antibody, and a highly toxic payload. For the effective delivery of such cytotoxic conjugates, a molecular target on the cancer cell is required. Various proteins are exclusively or abundantly expressed in cancer cells, making them a possible target for drug carriers. Fibroblast growth factor receptor 1 (FGFR1) overexpression has been reported in different types of cancer, but no FGFR1-targeting cytotoxic conjugate has been approved for therapy so far. In this study, the FGFR1-targeting peptide previously described in the literature was reformatted into a peptibody-peptide fusion with the fragment crystallizable (Fc) domain of IgG1. PeptibodyC19 can be effectively internalized into FGFR1-overexpressing cells and does not induce cells' proliferation. The main challenge for its use as a cytotoxic conjugate is a cysteine residue located within the targeting peptide. A standard drug-conjugation strategy based on the maleimide-thiol reaction involves modification of cysteines within the Fc domain hinge region. Applied here, however, may easily result in the modification of the targeting peptide with the drug, limiting its affinity to the target and therefore the potential for specific drug delivery. To investigate if this is the case, we have performed conjugation reactions with different auristatin derivatives (PEGylated and unmodified) under various conditions. By controlling the reduction conditions and the type of cytotoxic payload, different numbers of cysteines were substituted, allowing us to avoid conjugating the drug to the targeting peptide, which could affect its binding to FGFR1. The optimized protocol with PEGylated auristatin yielded doubly substituted peptibodyC19, showing specific cytotoxicity toward the FGFR1-expressing lung cancer cells, with no effect on cells with low FGFR1 levels. Indeed, additional cysteine poses a risk of unwanted modification, but changes in the type of cytotoxic payload and reaction conditions allow the use of standard thiol-maleimide-based conjugation to achieve standard Fc hinge region cysteine modification, analogously to antibody-drug conjugates.

A combined biochemical and cellular approach reveals Zn2+-dependent hetero- and homodimeric CD4 and Lck assemblies in T cells.

The CD4 or CD8 co-receptors' interaction with the protein-tyrosine kinase Lck initiates the tyrosine phosphorylation cascade leading to T cell activation. A critical question is: to what extent are co-receptors and Lck coupled? Our contribution concerns Zn2+, indispensable for CD4- and CD8-Lck formation. We combined biochemical and cellular approaches to show that dynamic fluctuations of free Zn2+ in physiological ranges influence Zn(CD4)2 and Zn(CD4)(Lck) species formation and their ratio, although the same Zn(Cys)2(Cys)2 cores. Moreover, we demonstrated that the affinity of Zn2+ to CD4 and CD4-Lck species differs significantly. Increased intracellular free Zn2+ concentration in T cells causes higher CD4 partitioning in the plasma membrane. We additionally found that CD4 palmitoylation decreases the specificity of CD4-Lck formation in the reconstituted membrane model. Our findings help elucidate co-receptor-Lck coupling stoichiometry and demonstrate that intracellular free Zn2+ has a major role in the interplay between CD4 dimers and CD4-Lck assembly.

From methodological limitations to the function of metallothioneins - a guide to approaches for determining weak, moderate, and tight affinity zinc sites.

Mammalian metallothioneins (MTs) are small cysteine-rich proteins whose primary role is participation in zinc and copper homeostasis. Ever since their discovery, MTs have been investigated in terms of metal-binding affinity. The initial concept of seven Zn(II) ions (Zn7MT) bound with the same, undifferentiated low-picomolar affinity in the α and ß domains prevailed for many years and derived from spectroscopic studies. The application of fluorescent zinc probes has changed the perception of MTs, showing that they function in nanomolar to subnanomolar free zinc concentrations due to the presence of tight, moderate, and weak binding sites. The discovery of Zn(II)-depleted MTs in many tissues and determination of cellular free Zn(II) concentrations with differentiated zinc affinity sites revealed the critical importance of partially saturated Zn4-6MTs species in cellular zinc buffering in a wide picomolar to nanomolar range of free Zn(II) concentrations. Until today, there was no clear agreement on the presence of differentiated or only tight zinc sites. Here, we present a series of spectroscopic, mass spectrometry-based, and enzymatic competition experiments that reveal how weak, moderate, or high-affinity ligands interact with human MT2, with special attention to the determination of Zn(II) affinities. The results show that the simplification of the stability model is the major reason for determining significantly different stability data that obscured the actual MTs function. Therefore, we emphasize that different metal affinities are the single most important reason for their presumed function, which changed over the years from tight binding and, thus, storage to one that is highly dynamic.

Deploying synthetic coevolution and machine learning to engineer protein-protein interactions.

Fine-tuning of protein-protein interactions occurs naturally through coevolution, but this process is difficult to recapitulate in the laboratory. We describe a platform for synthetic protein-protein coevolution that can isolate matched pairs of interacting muteins from complex libraries. This large dataset of coevolved complexes drove a systems-level analysis of molecular recognition between Z domain-affibody pairs spanning a wide range of structures, affinities, cross-reactivities, and orthogonalities, and captured a broad spectrum of coevolutionary networks. Furthermore, we harnessed pretrained protein language models to expand, in silico, the amino acid diversity of our coevolution screen, predicting remodeled interfaces beyond the reach of the experimental library. The integration of these approaches provides a means of simulating protein coevolution and generating protein complexes with diverse molecular recognition properties for biotechnology and synthetic biology.

Structural zinc binding sites shaped for greater works: Structure-function relations in classical zinc finger, hook and clasp domains.

Metal ions are essential elements present in biological systems able to facilitate many cellular processes including proliferation, signaling, DNA synthesis and repair. Zinc ion (Zn(II)) is an important cofactor for numerous biochemical reactions. Commonly, structural zinc sites demonstrate high Zn(II) affinity and compact architecture required for sequence-specific macromolecule binding. However, how Zn(II)-dependent proteins fold, how their dissociation occurs, and which factors modulate zinc protein affinity as well as stability remains not fully understood. The molecular rules governing precise regulation of zinc proteins function are hidden in the relationship between sequence and structure, and hence require deep understanding of their folding mechanism under metal load, reactivity and metal-to-protein affinity. Even though, this sequence-structure relationship has an impact on zinc proteins function, it has been shown that other biological factors including cellular localization and Zn(II) availability influence overall protein behavior. Taking into account all of the mentioned factors, in this review, we aim to describe the relationship between structure-function-stability of zinc structural sites, found in a zinc finger, zinc hook and zinc clasps, and reach far beyond a structural point of view in order to appreciate the balance between chemistry and biology that govern the protein world.

Zinc clasp-based reversible toolset for selective metal-mediated protein heterodimerization.

Considering the complex biological quandaries of the tightly woven networks of biological macromolecules, we present an optimized zinc clasp-based toolset from the CD4 co-receptor and Lck protein tyrosine kinase complex for selective, tight and fully reversible protein heterodimerization (log K12 = 18.6). We demonstrated its utility on CD4-tagged proteins with capture from bacterial lysate and constructed molecular baits using a new small-molecule tether.

Interdependence of free zinc changes and protein complex assembly - insights into zinc signal regulation.

Cellular zinc (Zn(ii)) is bound with proteins that are part of the proteomes of all domains of life. It is mostly utilized as a catalytic or structural protein cofactor, which results in a vast number of binding architectures. The Zn(ii) ion is also important for the formation of transient protein complexes with a Zn(ii)-dependent quaternary structure that is formed upon cellular zinc signals. The mechanisms by which proteins associate with and dissociate from Zn(ii) and the connection with cellular Zn(ii) changes remain incompletely understood. In this study, we aimed to examine how zinc protein domains with various Zn(ii)-binding architectures are formed under free Zn(ii) concentration changes and how formation of the Zn(ii)-dependent assemblies is related to the protein concentration and reactivity. To accomplish these goals we chose four zinc domains with different Zn(ii)-to-protein binding stoichiometries: classical zinc finger (ZnP), LIM domain (Zn2P), zinc hook (ZnP2) and zinc clasp (ZnP1P2) folds. Our research demonstrated a lack of changes in the saturation level of intraprotein zinc binding sites, despite various peptide concentrations, while homo- and heterodimers indicated a concentration-dependent tendency. In other words, at a certain free Zn(ii) concentration, the fraction of a formed dimeric complex increases or decreases with subunit concentration changes. Secondly, even small or local changes in free Zn(ii) may significantly affect protein saturation depending on its architecture, function and subcellular concentration. In our paper, we indicate the importance of interdependence of free Zn(ii) availability and protein subunit concentrations for cellular zinc signal regulation.

Molar absorption coefficients and stability constants of Zincon metal complexes for determination of metal ions and bioinorganic applications.

Zincon (ZI) is one of the most common chromophoric chelating probes for the determination of Zn2+ and Cu2+ ions. It is also known to bind other metal ions. However, literature data on its binding properties and molar absorption coefficients are rather poor, varying among publications or determined only in certain conditions. There are no systematic studies on Zn2+ and Cu2+ affinities towards ZI performed under various conditions. However, this widely commercially available and inexpensive agent is frequently the first choice probe for the measurement of metal binding and release as well as determination of affinity constants of other ligands/macromolecules of interest. Here, we establish the spectral properties and the stability of ZI and its complexes with Zn2+, Cu2+, Cd2+, Hg2+, Co2+, Ni2+ and Pb2+ at multiple pH values from 6 to 9.9. The obtained results show that in water solution the MZI complex is predominant, but in the case of Co2+ and Ni2+, M(ZI)2 complexes are also formed. The molar absorption coefficient at 618 nm for ZnZI and 599nm for CuZI complexes at pH7.4 in buffered (I=0.1M) water solutions are 24,200 and 26,100M-1cm-1, respectively. Dissociation constants of those complexes are 2.09×10-6 and 4.68×10-17M. We also characterized the metal-assisted Zincon decomposition. Our results provide new and reassessed optical and stability data that are applicable to a wide range of chemical and bioinorganic applications including metal ion detection, and quantification and affinity studies of ligands of interest. SYNOPSIS: Accurate values of molar absorption coefficients of Zincon complex with Zn2+, Cd2+, Hg2+, Co2+, Ni2+, Cu2+, and Pb2+ for rapid metal ion quantification are provided. Zincon stability constants with Zn2+ and Cu2+ in a wide pH range were determined.

Metal-coupled folding as the driving force for the extreme stability of Rad50 zinc hook dimer assembly.

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.

Molar absorption coefficients and stability constants of metal complexes of 4-(2-pyridylazo)resorcinol (PAR): Revisiting common chelating probe for the study of metalloproteins.

4-(2-Pyridylazo)resorcinol (PAR) is one of the most popular chromogenic chelator used in the determination of the concentrations of various metal ions from the d, p and f blocks and their affinities for metal ion-binding biomolecules. The most important characteristics of such a sensor are the molar absorption coefficient and the metal-ligand complex dissociation constant. However, it must be remembered that these values are dependent on the specific experimental conditions (e.g. pH, solvent components, and reactant ratios). If one uses these values to process data obtained in different conditions, the final result can be under- or overestimated. We aimed to establish the spectral properties and the stability of PAR and its complexes accurately with Zn(2+), Cd(2+), Hg(2+), Co(2+), Ni(2+), Cu(2+), Mn(2+) and Pb(2+) at a multiple pH values. The obtained results account for the presence of different species of metal-PAR complexes in the physiological pH range of 5 to 8 and have been frequently neglected in previous studies. The effective molar absorption coefficient at 492 nm for the ZnHx(PAR)2 complex at pH7.4 in buffered water solution is 71,500 M(-1) cm(-1), and the dissociation constant of the complex in these conditions is 7.08×10(-13) M(2). To confirm these values and estimate the range of the dissociation constants of zinc-binding biomolecules that can be measured using PAR, we performed several titrations of zinc finger peptides and zinc chelators. Taken together, our results provide the updated parameters that are applicable to any experiment conducted using inexpensive and commercially available PAR.