Evidence of variable human Fcγ receptor-Fc affinities across differentially-complexed IgG.

Antibody-mediated effector functions are widely considered to unfold according to an associative model of IgG-Fcγ receptor (FcγR) interactions. The associative model presupposes that Fc receptors cannot discriminate antigen-bound IgG from free IgG in solution and have equivalent affinities for each. Therefore, the clustering of Fcγ receptors (FcγR) in the cell membrane, cross-activation of intracellular signaling domains, and the formation of the immune synapse are all the result of avid interactions between the Fc region of IgG and FcγRs that collectively overcome the individually weak, transient interactions between binding partners. Antibody allostery, specifically conformational allostery, is a competing model in which antigen-bound antibody molecules undergo a physical rearrangement that causes them to stand out from the background of free IgG by virtue of greater FcγR affinity. Various evidence exists in support of this model of antibody allostery, but it remains controversial. We report observations from multiplexed, label-free kinetic experiments in which the affinity values of FcγR were characterized for covalently immobilized, captured, and antigen-bound IgG. Across the strategies tested, receptors had greater affinity for the antigen-bound mode of IgG presentation. This phenomenon was observed across multiple FcγRs and generalized to multiple antigens, antibody specificities, and subclasses. Furthermore, the thermodynamic signatures of FcγR binding to free or immune-complexed IgG in solution differed when measured by an orthogonal label-free method, but the failure to recapitulate the trend in overall affinity leaves open questions as to what additional factors may be at play.

Metallothionein-3 attenuates the effect of Cu2+ ions on actin filaments.

Metallothionein 3 (MT-3) is a cysteine-rich metal-binding protein that is expressed in the mammalian central nervous system and kidney. Various reports have posited a role for MT-3 in regulating the actin cytoskeleton by promoting the assembly of actin filaments. We generated purified, recombinant mouse MT-3 of known metal compositions, either with zinc (Zn), lead (Pb), or copper/zinc (Cu/Zn) bound. None of these forms of MT-3 accelerated actin filament polymerization in vitro, either with or without the actin binding protein profilin. Furthermore, using a co-sedimentation assay, we did not observe Zn-bound MT-3 in complex with actin filaments. Cu2+ ions on their own induced rapid actin polymerization, an effect that we attribute to filament fragmentation. This effect of Cu2+ is reversed by adding either EGTA or Zn-bound MT-3, indicating that either molecule can chelate Cu2+ from actin. Altogether, our data indicate that purified recombinant MT-3 does not directly bind actin but it does attenuate the Cu-induced fragmentation of actin filaments.

Metal binding and interdomain thermodynamics of mammalian metallothionein-3: enthalpically favoured Cu+ supplants entropically favoured Zn2+ to form Cu4 + clusters under physiological conditions.

Metallothioneins (MTs) are a ubiquitous class of small metal-binding proteins involved in metal homeostasis and detoxification. While known for their high affinity for d10 metal ions, there is a surprising dearth of thermodynamic data on metals binding to MTs. In this study, Zn2+ and Cu+ binding to mammalian metallothionein-3 (MT-3) were quantified at pH 7.4 by isothermal titration calorimetry (ITC). Zn2+ binding was measured by chelation titrations of Zn7MT-3, while Cu+ binding was measured by Zn2+ displacement from Zn7MT-3 with competition from glutathione (GSH). Titrations in multiple buffers enabled a detailed analysis that yielded condition-independent values for the association constant (K) and the change in enthalpy (ΔH) and entropy (ΔS) for these metal ions binding to MT-3. Zn2+ was also chelated from the individual α and ß domains of MT-3 to quantify the thermodynamics of inter-domain interactions in metal binding. Comparative titrations of Zn7MT-2 with Cu+ revealed that both MT isoforms have similar Cu+ affinities and binding thermodynamics, indicating that ΔH and ΔS are determined primarily by the conserved Cys residues. Inductively coupled plasma mass spectrometry (ICP-MS) analysis and low temperature luminescence measurements of Cu-replete samples showed that both proteins form two Cu4 +-thiolate clusters when Cu+ displaces Zn2+ under physiological conditions. Comparison of the Zn2+ and Cu+ binding thermodynamics reveal that enthalpically-favoured Cu+, which forms Cu4 +-thiolate clusters, displaces the entropically-favoured Zn2+. These results provide a detailed thermodynamic analysis of d10 metal binding to these thiolate-rich proteins and quantitative support for, as well as molecular insight into, the role that MT-3 plays in the neuronal chemistry of copper.

Mutant L-chain ferritins that cause neuroferritinopathy alter ferritin functionality and iron permeability.

In mammals, the iron storage and detoxification protein ferritin is composed of two functionally and genetically distinct subunit types, H (heavy) and L (light). The two subunits co-assemble in various ratios, with a tissue specific distribution, to form shell-like protein structures of 24 subunits within which a mineralized iron core is stored. The H-subunits possess ferroxidase centers that catalyze the rapid oxidation of ferrous ions, whereas the L-subunit does not have such centers and is believed to play an important role in electron transfer reactions that occur during the uptake and release of iron. Pathogenic mutations on the L-chain lead to neuroferritinopathy, a neurodegenerative disease characterized by abnormal accumulation of ferritin inclusion bodies and iron in the central nervous system. Here, we have characterized the thermal stability, iron loading capacity, iron uptake, and iron release properties of ferritin heteropolymers carrying the three pathogenic L-ferritin mutants (L154fs, L167fs, and L148fs, which for simplicity we named Ln1, Ln2 and Ln3, respectively), and a non-pathogenic variant (L135P) bearing a single substitution on the 3-fold axes of L-subunits. The UV-Vis data show a similar iron loading capacity (ranging between 1800 to 2400 Fe(iii)/shell) for all ferritin samples examined in this study, with Ln2 holding the least amount of iron (i.e. 1800 Fe(iii)/shell). The three pathogenic L-ferritin mutants revealed higher rates of iron oxidation and iron release, suggesting that a few mutated L-chains on the heteropolymer have a significant effect on iron permeability through the ferritin shell. DSC thermograms showed a strong destabilization effect, the severity of which depends on the location of the frameshift mutations (i.e. wt heteropolymer ferritin ≅ homopolymer H-chain > L135P > Ln2 > Ln1 > Ln3). Variant L135P had only minor effects on the protein functionality and stability, suggesting that local melting of the 3-fold axes in this variant may not be responsible for neuroferritinopathy-like disorders. The data support the hypothesis that hereditary neuroferritinopathies are due to alterations of ferritin functionality and lower physical stability which correlate with the frameshifts introduced at the C-terminal sequence and explain the dominant transmission of the disorder.

The Lysosomal Protein Saposin†B Binds Chloroquine.

Chloroquine (CQ) has been widely used in the treatment of malaria since the 1950s, though toxicity and resistance is increasingly limiting its use in the clinic. More recently, CQ is also becoming recognized as an important therapeutic compound for the treatment of autoimmune disorders and has shown activity as an anticancer agent. However, the full extent of CQ pharmacology in humans is still unclear. Herein, we demonstrate that the lysosomal protein saposin B (sapB), critical for select lipid degradation, binds CQ with implications for both CQ function and toxicity. Using isothermal titration calorimetry (ITC) and fluorescence quenching experiments, CQ was shown to bind to the dimeric form of sapB at both pH 5.5 and pH 7.4 with an average binding affinity of 2.3×10(4) m(-1). X-ray crystallography confirmed this, and the first complete crystal structure of sapB with a bound small molecule (CQ) is reported. The results suggest that sapB might play a role in mitigating CQ-based toxicity and that sapB might itself be overwhelmed by CQ causing impaired lipid degradation.