Flexibility of the CueR Metal Site Probed by Instantaneous Change of Element and Oxidation State from Ag^I to Cd^II

Abstract

Selectivity for monovalent metal ions is an important facet of the function of the metalloregulatory protein CueR. 111Ag perturbed angular correlation of γ‐rays (PAC) spectroscopy probes the metal site structure and the relaxation accompanying the instantaneous change from AgI to CdII upon 111Ag radioactive decay. That is, a change from AgI, which activates transcription, to CdII, which does not. In the frozen state (−196 °C) two nuclear quadrupole interactions (NQIs) are observed; one (NQI1) agrees well with two coordinating thiolates and an additional longer contact to the S77 backbone carbonyl, and the other (NQI2) reflects that CdII has attracted additional ligand(s). At 1 °C only NQI2 is observed, demonstrating that relaxation to this structure occurs within ≈10 ns of the decay of 111Ag. Thus, transformation from AgI to CdII rapidly disrupts the functional linear bis(thiolato)AgI metal site structure. This inherent metal site flexibility may be central to CueR function, leading to remodelling into a non‐functional structure upon binding of non‐cognate metal ions. In a broader perspective, 111Ag PAC spectroscopy may be applied to probe the flexibility of protein metal sites.

Tying Up a Loose End: On the Role of the C‐Terminal CCHHRAG Fragment of the Metalloregulator CueR

“…At 1 °C, the 111 Ag PAC spectrum of WT‐CueR is relatively simple, reflecting only one NQI which is very similar to the low frequency component observed at −196 °C [18] (Figure 3). Thus, under these conditions, the metal site relaxes to a well‐defined coordination geometry with a coordination number higher than 2 [18] within about 10 ns after the decay.…”

“…At 1 °C, the 111 Ag PAC spectrum of WT‐CueR is relatively simple, reflecting only one NQI which is very similar to the low frequency component observed at −196 °C [18] (Figure 3). Thus, under these conditions, the metal site relaxes to a well‐defined coordination geometry with a coordination number higher than 2 [18] within about 10 ns after the decay. Interestingly, the same signal is observed for the Δ7C‐CueR variant, but it only accounts for ∼29 % of the total signal amplitude, and a lower frequency signal with massive line broadening is also present.…”

“…One is a high frequency signal, NQI 1 , which reflects the original bisthiolato‐Ag I coordination geometry having undergone minor relaxation upon the decay to Cd II , presumably leading to an additional weak interaction with the carbonyl oxygen of Ser77. The other, NQI 2 , is a lower frequency signal, reflecting higher coordination number than 2 [18,29] . The Δ7C‐CueR variant exhibits essentially the same high frequency signal, NQI 1 (see Figure 3 and Table 1), demonstrating that this native Ag I ‐CueR metal site coordination geometry is the same as in the WT protein.…”

“…The change of element and oxidation state, and the recoil energy shifts the system out of equilibrium, and in the subsequent relaxation process Cd II explores the local potential energy surface. Thus, the 111 Ag PAC signal provides information on both the metal site structure and local rigidity/flexibility [18,26–28] …”

“…In our previous work with peptide models and the full length CueR protein we have shown that the metal binding loop of CueR is flexible enough to accommodate non‐cognate divalent metal ions, such as Hg II and Cd II , in different coordination geometries [15–18] . However, these non‐cognate metal ions may disorganize the structure of the metal binding region and thereby presumably hinder the function of the protein [18] .…”