Abolishing "structural blindness" in metalloproteins: PioC, a NOE-less protein structure

Almost half of all known enzymes are metalloproteins where the metal center(s) are essential for catalysis, electron transfer, metal storage/transport, or provide stability and structural properties. NMR is a privileged method for characterizing metalloproteins providing the structure at atomic resolution, information about amplitude and timescale of internal dynamics, hints on electronic structure and oxidation states in conditions that mimic the physiological context. However, in a significant part of the metalloproteome the metal ion is paramagnetic and in its vicinity, a “blind sphere” exists where nuclear relaxation is enhanced and signal detection becomes a challenge. This challenge may be circumvented by substituting the paramagnetic metal with a diamagnetic analogue. Yet, this strategy often fails since it leads to unfolded proteins or the diamagnetic analogue may not mimic adequately the native paramagnetic metal. Using recent developments in pulse sequences, here we present a strategy for achieving structure determination in paramagnetic proteins. PioC from Rhodopseudomonas palustris TIE1 is the smallest High Potential IronSulfur Protein (HiPIP) ever isolated. The paramagnetism from the [4Fe4S] cluster affects 60% of the protein, making it the perfect example of the dual nature of paramagnetic NMR. On one side relaxation precludes signal detection, and on the other it provides unique sets of information. The structure of PioC was determined by NMR using two different sets of restraints, one containing Nuclear Overhauser Enhancements (NOEs) and another containing Paramagnetic Relaxation Enhancements (PREs). These were used independently and then combined revealing that under favorable conditions, PREs can efficiently complement and eventually replace NOEs for structural characterization.