The order of PDZ3 and TrpCage in fusion chimeras determines their properties-a biophysical characterization

Abstract

Most of the structural proteins known today are composed of domains that carry their own functions while keeping their structural properties. It is supposed that such domains, when taken out of the context of the whole protein, can retain their original structure and function to a certain extent. Information on the specific functional and structural characteristics of individual domains in a new context of artificial fusion proteins may help to reveal the rules of internal and external domain communication. Moreover, this could also help explain the mechanism of such communication and address how the mutual allosteric effect plays a role in a such multi-domain protein system. The simple model system of the two-domain fusion protein investigated in this work consisted of a well-folded PDZ3 domain and an artificially designed small protein domain called Tryptophan Cage (TrpCage). Two fusion proteins with swapped domain order were designed to study their structural and functional features as well as their biophysical properties. The proteins composed of PDZ3 and TrpCage, both identical in amino acid sequence but different in composition (PDZ3-TrpCage, TrpCage-PDZ3), were studied using circualr dichroism (CD) spectrometry, analytical ultracentrifugation, and molecular dynamic simulations. The biophysical analysis uncovered different structural and denaturation properties of both studied proteins, revealing their different unfolding pathways and dynamics.

Keywords: chimeras; fusion protein; protein domains; protein dynamic studies.

FIGURE 1

PDZ3 present in MAGUK tandem of ZO‐1. ZO‐1 protein consists of three PDZ domains (PDZ1 and PDZ2 dark pink, PDZ3 light pink), SH3 domain (red) and GUK domain (green), PDB 3TSZ. PDZ3 domain occurs as a first member of PDZ3‐SH3‐GUK supramodule. PDZ3 and SH3 domains are linked by an α‐helical linker, which enhances the binding affinity of PDZ3 to the active binding partners

FIGURE 2

FD3A and FD4A design and biochemical characterization. (a) PDZ3 (pink) from ZO‐1 structure (PDB:3SHU). The core of the domain consists of six β‐sheet strands (βA–βE) surrounded by two α‐helices (αA–αB). (b) TrpCage (cyan) structure (PDB:1L2Y) composed of α‐helix and poly‐proline helix which keeps the structure compact. (c) Schematic representation of FD3A and FD4A fusion proteins design. PDZ3 domain (pink) was fused with α‐helical mini‐protein TrpCage (cyan) in forward (FD3A: PZ3‐TrpCage, labeled by red) and reverse (FD4A: TrpCage‐PDZ3, labeled by blue) orders. The domains were fused by a flexible linker (gray) in order to keep domain structure individuality and promote a possible interdomain communication. (d) The size exclusion chromatography (SEC) profile of FD3A (red) and FD4A (blue) indicates slightly lower compactness of FD4A compared to FD3A. (e) Dynamic light scattering (DLS) spectra of FD3A (red) and FD4A (blue) confirmed the same trend of distinct hydrodynamic radii between FD3A and FD4A. (f) Comparison of DLS estimated hydrodynamic radii (Rh), polydispersity indexes (PDI) and predicted molecular weight (M_W) of FD3A and FD4A. The measurements were performed 20x for each sample, and final values represent the mean value with SD

FIGURE 3

FD3A and FD4A are mostly monomeric in solution. (a) Overlay of the fitted c(s) continuous size distributions of the sedimenting species for FD3A (left panel) and FD4A (right panel at concentrations ranging from 6 to 120 μM. While FD3A is a monomer in the whole concentration range tested, FD4A showed a monomer‐dimer equilibrium from 30 μM concentration. (b) Summary of the sedimentation coefficients (s_20,w), frictional ratios (f/f₀), Stokes hydrodynamic radii (Rh), and molecular weights (M_W; estimated from the fitted s_20,w and f/f₀ values) obtained from AUC data for FD3A and FD4A chimeras

FIGURE 4

Thermal unfolding of FD3A and FD4A fusion proteins. (a) Far‐UV CD spectra of FD3A, FD4A, PDZ3, and TrpCage expressed as molar ellipticity as a function of wavelength. (b) Near‐UV spectra of FD3A and FD4A expressed as molar ellipticity as a function of wavelength. (c) The thermal unfolding of FD3A (red) and FD4A (blue) fusion proteins and PDZ3 (pink) and TrpCage (cyan) domains expressed as dependence of molar ellipticity on temperature at 224 nm. (d) Summary of melting temperatures (Tm) of FD3A (red) and FD4A (blue) fusion proteins and PDZ3 (pink) and TrpCage (cyan) domains

FIGURE 5

The urea‐induced unfolding of FD3A and FD4A fusion proteins, and of PDZ3 and TrpCage individual domains. Far‐UV CD spectra of (a) FD3A, (b) FD4A, (c) PDZ3 and (d) TrpCage were measured over the 0.0–6.0 M urea concentration range with increment of 0.5 M. The CD spectra were expressed as molar ellipticities at 222 nm as a function of molar concentrations of urea

FIGURE 6

Molecular dynamics simulations of FD3A and FD4A molecular models. The RMSD analysis of selected 200 ns molecular dynamic simulations (MDs) of (a) FD3A, (b) FD4A closed state and (c) FD4A open state. Root mean square deviation (RMSD) of C‐α atoms were analysed separately for TrpCage and PDZ3 domain to confirm the structural stability of the individual domains during molecular dynamic simulations (MDs). The structural stabilities of the whole molecules were analysed as RMSD of selected C‐α atoms corresponding to regions with defined secondary structure in the initial model. In all cases, RMSD is determined by comparing desired frame of MDs runs to the relevant initial structure. In the case of bottom right plot (C) for FD4A open state, the gray line shows RMSD computed for open conformation with compact conformation as a reference structure. The initial structures of (d) FD3A, (e) FD4A closed state and (f) FD4A open state consisting of PDZ3 domain and Trp‐cage (PDZ3 domain is shown in pink, TrpCage is shown in cyan, linker region and terminal extensions are shown in gray)