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Methods for Solving Highly Symmetric De Novo Designed Metalloproteins: Crystallographic Examination of a Novel Three-Stranded Coiled-Coil Structure Containing d-Amino Acids.

ABSTRACT: The core objective of de novo metalloprotein design is to define metal-protein relationships that control the structure and function of metal centers by using simplified proteins. An essential requirement to achieve this goal is to obtain high resolution structural data using either NMR or crystallographic studies in order to evaluate successful design. X-ray crystal structures have proven that a four heptad repeat scaffold contained in the three-stranded coiled coil (3SCC), called CoilSer (CS), provides an excellent motif for modeling a three Cys binding environment capable of chelating metals into geometries that resemble heavy metal sites in metalloregulatory systems. However, new generations of more complicated designs that feature, for example, a d-amino acid or multiple metal ligand sites in the helical sequence require a more stable construct. In doing so, an extra heptad was introduced into the original CS sequence, yielding a GRAND-CoilSer (GRAND-CS) to retain the 3SCC folding. An apo-(GRAND-CSL12DLL16C)3 crystal structure, designed for Cd(II)S3 complexation, proved to be a well-folded parallel 3SCC. Because this structure is novel, protocols for crystallization, structural determination, and refinements of the apo-(GRAND-CSL12DLL16C)3 are described. This report should be generally useful for future crystallographic studies of related coiled-coil designs.

SUBMITTER: Ruckthong L

PROVIDER: S-EPMC5237576 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Methods for Solving Highly Symmetric De Novo Designed Metalloproteins: Crystallographic Examination of a Novel Three-Stranded Coiled-Coil Structure Containing d-Amino Acids.

Ruckthong L L Stuckey J A JA Pecoraro V L VL

Methods in enzymology 20160623

The core objective of de novo metalloprotein design is to define metal-protein relationships that control the structure and function of metal centers by using simplified proteins. An essential requirement to achieve this goal is to obtain high resolution structural data using either NMR or crystallographic studies in order to evaluate successful design. X-ray crystal structures have proven that a four heptad repeat scaffold contained in the three-stranded coiled coil (3SCC), called CoilSer (CS), ...[more]

PMID: 27586331

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Project description:Herein we report how de novo designed peptides can be used to investigate whether the position of a metal site along a linear sequence that folds into a three-stranded α-helical coiled coil defines the physical properties of Cd(II) ions in either CdS(3) or CdS(3)O (O-being an exogenous water molecule) coordination environments. Peptides are presented that bind Cd(II) into two identical coordination sites that are located at different topological positions at the interior of these constructs. The peptide GRANDL16PenL19IL23PenL26I binds two Cd(II) as trigonal planar 3-coordinate CdS(3) structures whereas GRANDL12AL16CL26AL30C sequesters two Cd(II) as pseudotetrahedral 4-coordinate CdS(3)O structures. We demonstrate how for the first peptide, having a more rigid structure, the location of the identical binding sites along the linear sequence does not affect the physical properties of the two bound Cd(II). However, the sites are not completely independent as Cd(II) bound to one of the sites ((113)Cd NMR chemical shift of 681 ppm) is perturbed by the metalation state (apo or [Cd(pep)(Hpep)(2)](+) or [Cd(pep)(3)](-)) of the second center ((113)Cd NMR chemical shift of 686 ppm). GRANDL12AL16CL26AL30C shows a completely different behavior. The physical properties of the two bound Cd(II) ions indeed depend on the position of the metal center, having pK(a2) values for the equilibrium [Cd(pep)(Hpep)(2)](+) → [Cd(pep)(3)](-) + 2H(+) (corresponding to deprotonation and coordination of cysteine thiols) that range from 9.9 to 13.9. In addition, the L26AL30C site shows dynamic behavior, which is not observed for the L12AL16C site. These results indicate that for these systems one cannot simply assign a "4-coordinate structure" and assume certain physical properties for that site since important factors such as packing of the adjacent Leu, size of the intended cavity (endo vs exo) and location of the metal site play crucial roles in determining the final properties of the bound Cd(II).

Project description:An important factor that defines the toxicity of elements such as cadmium(II), mercury(II), and lead(II) with biological macromolecules is metal ion exchange dynamics. Intriguingly, little is known about the fundamental rates and mechanisms of metal ion exchange into proteins, especially helical bundles. Herein, we investigate the exchange kinetics of Cd(II) using de novo designed three-stranded coiled-coil peptides that contain metal complexing cysteine thiolates as a model for the incorporation of this ion into trimeric, parallel coiled coils. Peptides were designed containing both a single Cd(II) binding site, GrandL12AL16C [Grand = AcG-(LKALEEK)(5)-GNH(2)], GrandL26AL30C, and GrandL26AE28QL30C, as well as GrandL12AL16CL26AL30C with two Cd(II) binding sites. The binding of Cd(II) to any of these sites is of high affinity (K(A) > 3 × 10(7) M(-1)). Using (113)Cd NMR spectroscopy, Cd(II) binding to these designed peptides was monitored. While the Cd(II) binding is in extreme slow exchange regime without showing any chemical shift changes, incremental line broadening for the bound (113)Cd(II) signal is observed when excess (113)Cd(II) is titrated into the peptides. Most dramatically, for one site, L26AL30C, all (113)Cd(II) NMR signals disappear once a 1.7:1 ratio of Cd(II)/(peptide)(3) is reached. The observed processes are not compatible with a simple "free-bound" two-site exchange kinetics at any time regime. The experimental results can, however, be simulated in detail with a multisite binding model, which features additional Cd(II) binding site(s) which, once occupied, perturb the primary binding site. This model is expanded into differential equations for five-site NMR chemical exchange. The numerical integration of these equations exhibits progressive loss of the primary site NMR signal without a chemical shift change and with limited line broadening, in good agreement with the observed experimental data. The mathematical model is interpreted in molecular terms as representing binding of excess Cd(II) to surface Glu residues located at the helical interfaces. In the absence of Cd(II), the Glu residues stabilize the three-helical structure though salt bridge interactions with surface Lys residues. We hypothesize that Cd(II) interferes with these surface ion pairs, destabilizing the helical structure, and perturbing the primary Cd(II) binding site. This hypothesis is supported by the observation that the Cd(II)-excess line broadening is attenuated in GrandL26AE28QL30C, where a surface Glu(28), close to the metal binding site, was changed to Gln. The external binding site may function as an entry pathway for Cd(II) to find its internal binding site following a molecular rearrangement which may serve as a basis for our understanding of metal complexation, transport, and exchange in complex native systems containing ?-helical bundles.

Dataset Information

Methods for Solving Highly Symmetric De Novo Designed Metalloproteins: Crystallographic Examination of a Novel Three-Stranded Coiled-Coil Structure Containing d-Amino Acids.

Publications

Methods for Solving Highly Symmetric De Novo Designed Metalloproteins: Crystallographic Examination of a Novel Three-Stranded Coiled-Coil Structure Containing d-Amino Acids.

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