In†Cellulo Mössbauer and EPR Studies Bring New Evidence to the Long-Standing Debate on Iron-Sulfur Cluster Binding in Human Anamorsin.

Human anamorsin is an iron-sulfur (Fe-S)-cluster-binding protein acting as an electron donor in the early steps of cytosolic iron-sulfur protein biogenesis. Human anamorsin belongs to the eukaryotic CIAPIN1 protein family and contains two highly conserved cysteine-rich motifs, each binding an Fe-S cluster. In vitro works by various groups have provided rather controversial results for the type of Fe-S clusters bound to the CIAPIN1 proteins. In order to unravel the knot on this topic, we used an in cellulo approach combining Mössbauer and EPR spectroscopies to characterize the iron-sulfur-cluster-bound form of human anamorsin. We found that the protein binds two [2Fe-2S] clusters at both its cysteine-rich motifs.

Unraveling the mechanism of [4Fe-4S] cluster assembly on the N-terminal cluster binding site of NUBP1.

[4Fe-4S]2+ cluster assembly in human cytosol requires both a [2Fe-2S] cluster chaperone being able to donate two [2Fe-2S]2+ clusters and an electron donor providing two electrons to reductively couple the two [2Fe-2S]2+ clusters into a [4Fe-4S]2+ cluster. The mechanism through which the cytosolic [4Fe-4S]2+ cluster assembly works is still not defined. Here, we show that a hetero-tetrameric complex formed by two molecules of cluster-reduced [2Fe-2S]+ 2 -anamorsin and one molecule of dimeric cluster-oxidized [2Fe-2S]2+ 2 -GLRX32 orchestrates the assembly of a [4Fe-4S]2+ cluster on the N-terminal cluster binding site of the cytosolic protein NUBP1. We demonstrate that the hetero-tetrameric complex is able to synergically provide two [2Fe-2S]2+ clusters from GLRX3 and two electrons from anamorsin for the assembly of the [4Fe-4S]2+ cluster on the N-terminal cluster binding site of NUBP1. We also showed that only one of the two [2Fe-2S] clusters bound to anamorsin, that is, that bound to the CX8 CX2 CXC motif, provides the electrons required to form the [4Fe-4S]2+ cluster. Our study contributes to the molecular understanding of the mechanism of [4Fe-4S] protein biogenesis in the cytosol.

A novel method using nuclear magnetic resonance for plasma protein binding assessment in drug discovery programs.

A new methodology based on Nuclear Magnetic Resonance (NMR) was developed to determine plasma protein binding (PPB) of drug candidates in drug discovery programs. A strong correlation was found between the attenuation of NMR signals of diverse drugs in the presence of different plasma concentrations and their fraction bound (fb) reported in the literature. Based on these results, a protocol for a rapid calculation of fb of small molecules was established. The advantage of using plasma instead of purified recombinant proteins and the possibility of pool analysis to increase throughput were also evaluated. This novel methodology proved to be very versatile, cost-effective, fast and suitable for automation. As a plus, it contemporarily provides a quality check and solubility of the compound.