Characterizing metal-binding sites in proteins with X-ray crystallography

Abstract

Metals have crucial roles in many physiological, pathological, toxicological, pharmaceutical, and diagnostic processes. Proper handling of metal-containing macromolecule samples for structural studies is not trivial, and failure to handle them properly is often a source of irreproducibility caused by issues such as pH changes, incorporation of unexpected metals, or oxidization/reduction of the metal. This protocol outlines the guidelines and best practices for characterizing metal-binding sites in protein structures and alerts experimenters to potential pitfalls during the preparation and handling of metal-containing protein samples for X-ray crystallography studies. The protocol features strategies for controlling the sample pH and the metal oxidation state, recording X-ray fluorescence (XRF) spectra, and collecting diffraction data sets above and below the corresponding metal absorption edges. This protocol should allow experimenters to gather sufficient evidence to unambiguously determine the identity and location of the metal of interest, as well as to accurately characterize the coordinating ligands in the metal binding environment within the protein. Meticulous handling of metal-containing macromolecule samples as described in this protocol should enhance experimental reproducibility in biomedical sciences, especially in X-ray macromolecular crystallography. For most samples, the protocol can be completed within a period of 7-190 d, most of which (2-180 d) is devoted to growing the crystal. The protocol should be readily understandable to structural biologists, particularly protein crystallographers with an intermediate level of experience.

Encapsulation of the Dinuclear Trithiolato‐Bridged Arene Ruthenium Complex Diruthenium‐1 in an Apoferritin Nanocage: Structure and Cytotoxicity

“…To further confirm the presence of Ru atoms within the crystals of DiRu‐1‐encapsulated AFt, ICP‐MS measurements were performed. Similar experiments were performed by Rompel and co‐workers to verify the presence of KP1019 in human serum albumin–KP1019 crystals and also suggested in recent protocols for the determination of the X‐ray structures of the adducts formed in the reaction between proteins and metallodrugs . Crystals of AFt and of DiRu‐1‐encapsulated AFt were extensively washed with the reservoir solution and then dissolved in 20 μL of milli‐Q water.…”

“…Similar experiments were performed by Rompel and co-workerst ov erify the presence of KP1019 in human serum albumin-KP1019 crystals [43] and also suggested in recent protocols for the determination of the X-ray structures of the adducts formed in the reaction between proteins and metallodrugs. [44,45] Crystals of AFt and of DiRu-1-encapsulated AFt were extensively washed with the reservoir solution and then dissolvedi n2 0mLo fm illi-Q water.T hese solutionsw erea nalyzed by ICP-MS;t he datai ndicate that AFt crystalsc ontained an undetectable amount of Ru, whereas the solution with DiRu-1-encapsulated AFt crystals contained as ignificant amount of Ru (37 ng). Altogether,t hese findings unambiguously demonstrate that DiRu-1 was successfully encapsulated within the AFt cage, that the structure of the protein in DiRu-1-encapsulated AFt is undistinguishable from AFt alone, and that DiRu-1 does not coordinate to the protein, but is trapped in the bulk.…”

A microplate screen to estimate metal-binding affinities of metalloproteins

“…Finally, the flow-through was applied to an FPLC with an anion exchange resin (MonoQ) to improve the protein purity and dialyzed once more with Chelex 100 for 24 hr to reduce the concentration of NaCl and any remaining metals. This whole procedure yielded CcSBPII at 2.2 mg/L of culture with a final concentration of 1 mg/mL with 0.5 ppb nickel, representing a 0.01 metal-protein ratio that is recommended by Handing et al 2018 and used in a study that measured metal binding affinity by ITC. 18,36 Prior to using CcSBPII in IPFQ-LTAs after thawing, the integrity of its secondary structures was evaluated using CD ( Figure 4C).…”

“…This whole procedure yielded CcSBPII at 2.2 mg/L of culture with a final concentration of 1 mg/mL with 0.5 ppb nickel, representing a 0.01 metal-protein ratio that is recommended by Handing et al 2018 and used in a study that measured metal binding affinity by ITC. 18,36 Prior to using CcSBPII in IPFQ-LTAs after thawing, the integrity of its secondary structures was evaluated using CD ( Figure 4C). We found that it maintained a primarily α-helical conformation, but a peak was missing at 220 nm that is typical of α-helical proteins.…”

STRIDER: Steric hindrance estimator

“…may require multiple conformers to overcome the sampling issues, STRIDER accepts ensemble of conformers and reports pairwise steric hindrance profile for individual conformers in an interactive manner. Although the metal ion coordination has an important role in biomolecular stability (Largy et al, 2016, Zhang et al, 2014, Bhattacharyya et al, 2016 and function (Riordan, 1977, Pattammattel et al, 2013, Liu et al, 2014, identifying metal ions directly by NMR and X-ray crystallography still remains a challenge (Jensen et al, 2005, Erat and Sigel, 2011, Nabuurs et al, 2006, Handing et al, 2018, thus, calls for metal modeling. Thus, when a query structure with metal ion is submitted to STRIDER to access the accuracy of the modeled metal coordination, it reports the metal coordination number and distance profile of metal with its coordinating partners in the binding pocket.…”