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The Maf protein family belongs to the activator protein 1 (AP-1) superfamily of transcription factors that bind specific DNA target sequences through a basic region and exploit a leucine zipper (LZ) motif for protein–protein interactions leading to homo- or hetero-dimerization. Mafs unique DNA-binding domain contains a highly conserved extended homology region (EHR) that allows to recognize longer DNA sequences than other basic leucine zipper (bZIP) transcription factors. Inspired by the fact that overexpression of Mafs is observed in about 50% of cases of multiple myeloma, a hematological malignant disorder, we undertook a peptide inhibitor approach. The LZ domain of c-Maf, one of large Mafs, was produced by solid phase peptide synthesis. We characterized its secondary structure and dimerization properties, and found that dimerization and folding events are strictly coupled. Moreover, potential peptidic c-Maf dimerization inhibitors were computationally designed and synthesized. These compounds were demonstrated by circular dichroism (CD) spectroscopy and MALDI-TOF mass spectrometry to bind to c-Maf LZ monomers, to drive folding of their partially disordered structure and to efficiently compete with dimerization, suggesting a way for interfering with the function of c-Maf and, more generally, of intrinsically disordered proteins, till now considered undruggable targets. [ABSTRACT FROM AUTHOR]

Abstract: Many fish hemoglobins exhibit a marked dependence of oxygen affinity and cooperativity on proton concentration, called Root effect. Both tertiary and quaternary effects have been evoked to explain the allosteric regulation brought about by protons in fish hemoglobins. However, no general rules have emerged so far. We carried out a complementary crystallographic and microspectroscopic characterization of ligand binding to crystals of deoxy-hemoglobin from the Antarctic fish Trematomus bernacchii (HbTb) at pH6.2 and pH8.4. At low pH ligation has negligible structural effects, correlating with low affinity and absence of cooperativity in oxygen binding. At high pH, ligation causes significant changes at the tertiary structural level, while preserving structural markers of the T state. These changes mainly consist in a marked displacement of the position of the switch region CD corner towards an R-like position. The functional data on T-state crystals validate the relevance of the crystallographic observations, revealing that, differently from mammalian Hbs, in HbTb a significant degree of cooperativity in oxygen binding is due to tertiary conformational changes, in the absence of the T–R quaternary transition. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins. [Copyright &y& Elsevier]

Abstract: In the last decade, protein allostery has experienced a major resurgence, boosted by the extension of the concept to systems of increasing complexity and by its exploitation for the development of drugs. Expansion of the field into new directions has not diminished the key role of hemoglobin as a test molecule for theory and experimental validation of allosteric models. Indeed, the diffusion of hemoglobins in all kingdoms of life and the variety of functions and of quaternary assemblies based on a common tertiary fold indicate that this superfamily of proteins is ideally suited for investigating the physical and molecular basis of allostery and firmly maintains its role as a main player in the field. This review is an attempt to briefly recollect common and different strategies adopted by metazoan hemoglobins, from monomeric molecules to giant complexes, exploiting homotropic and heterotropic allostery to increase their functional dynamic range. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins. [Copyright &y& Elsevier]

Abstract: Single crystal microspectrophotometry has emerged as a valuable technique for monitoring molecular events that take place within protein crystals, thus tightly coupling structure to function. Absorption and fluorescence spectra, ligand binding affinities and kinetic constants can be determined, allowing i) the definition of the experimental conditions for X-ray crystallography experiments and their interpretation, ii) the assessment of whether crystal lattice forces have altered conformational equilibria, iii) the comparison with data obtained in solution. Microspectrophotometric measurements using oriented crystals and linearly polarized light are carried out usually off-line with respect to X-ray data collection and are aimed at an in- depth characterization of protein function in the crystal, leading to robust structure–function relationships. The power of this approach is highlighted by reporting a few case studies, including hemoglobins, pyridoxal 5′-phosphate-dependent enzymes and acetylcholinesterases. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State. [Copyright &y& Elsevier]

Abstract: Pyridoxal 5’-phosphate (PLP) dependent methionine γ-lyase catalyzes the breakdown of L-methionine to α-ketobutyric acid, methanethiol and ammonia. This enzyme, present in anaerobic microorganisms, has biomedical interest both for its activity as antitumor agent, depleting methionine supply in methionine-dependent cancers, and as target in the treatment of human pathogen infections, activating the pro-drug trifluoromethionine. To validate the structure of the enzyme from Citrobacter freundii, crystallized from monomethyl ether polyethylene glycol 2000, for the development of lead compounds, the reactivity of the crystalline enzyme towards L-methionine, substrate analogs and inhibitors was determined by polarized absorption microspectrophotometry. Spectral data were also collected for enzyme crystals, grown in monomethyl ether polyethylene glycol 2000 in the presence of ammonium sulfate. The three-dimensional structure of these enzyme crystals, solved at 1.65Å resolution with Rfree 23.2%, revealed the surprising absence of the aldimine bond between the active site Lys210 and PLP. Different hypothesis are proposed and discussed in the light of spectral and structural data, pointing out to the relevance of the complementarity between X-ray crystallography and single crystal spectroscopy for the understanding of biological mechanisms at molecular level. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State. [Copyright &y& Elsevier]

Abstract: Historically, exogenous administration of hemoglobin solutions to implement the oxygen transport capacity for clinical applications suffered from dramatic drawbacks, resulting in the failure of many attempts. In the last decades, the biochemical and physiological basis responsible for the therapeutic failures has been extensively investigated. It is now widely accepted that they mostly arise because, out of the confined and controlled environment of the red blood cell, hemoglobin exhibits tetramer instability, increased auto-oxidation rate, higher oxygen affinity, altered cooperativity and nitric oxide reactivity. Moreover, it became evident that the design of a hemoglobin-based oxygen carrier that exactly reproduces the “physiological” oxygen-binding curve is not only an overly ambitious task, but may also represent a wrong approach for many potential clinical applications. Under these premises, and given the complex chemical nature of blood, it is obvious that any strategy undertaken to modify the stability and function of the hemoglobin tetramer for clinical use should be driven by a detailed knowledge of its structure, dynamics and mechanism of allosteric regulation. We briefly review the most recent theories and experiments that increased our understanding of the mechanism of homo- and heterotropic effects in human hemoglobin, trying to interpret, on a biophysical basis, how diverse approaches like polymerization, cross-linking, site-directed mutagenesis, surface decoration and encapsulation may affect ligand affinity and allosteric regulation. [Copyright &y& Elsevier]

Serine racemase is the pyridoxal 5′-phosphate dependent enzyme that catalyzes both production and catabolism of d -serine, a co-agonist of the NMDA glutamate receptors. Mg 2 + , or, alternatively, Ca 2 + , activate human serine racemase by binding both at a specific site and – as ATP-metal complexes – at a distinct ATP binding site. We show that Mg 2 + and Ca 2 + bind at the metal binding site with a 4.5-fold difference in affinity, producing a similar thermal stabilization and partially shifting the dimer-tetramer equilibrium in favour of the latter. The ATP-Ca 2 + complex produces a 2-fold lower maximal activation in comparison to the ATP-Mg 2 + complex and exhibits a 3-fold higher EC 50 . The co-presence of ATP and metals further stabilizes the tetramer. In consideration of the cellular concentrations of Mg 2 + and Ca 2 + , even taking into account the fluctuations of the latter, these results point to Mg 2 + as the sole physiologically relevant ligand both at the metal binding site and at the ATP binding site. The stabilization of the tetramer by both metals and ATP-metal complexes suggests a quaternary activation mechanism mediated by 5′-phosphonucleotides similar to that observed in the distantly related prokaryotic threonine deaminases. This allosteric mechanism has never been observed before in mammalian fold type II pyridoxal 5′-phosphate dependent enzymes. [ABSTRACT FROM AUTHOR]