Supertertiary structure of the synaptic MAGuK scaffold proteins is conserved

Abstract

Scaffold proteins form a framework to organize signal transduction by binding multiple partners within a signaling pathway. This shapes the output of signal responses as well as providing specificity and localization. The Membrane Associated Guanylate Kinases (MAGuKs) are scaffold proteins at cellular junctions that localize cell surface receptors and link them to downstream signaling enzymes. Scaffold proteins often contain protein-binding domains that are connected in series by disordered linkers. The tertiary structure of the folded domains is well understood, but describing the dynamic inter-domain interactions (the superteritary structure) of such multidomain proteins remains a challenge to structural biology. We used 65 distance restraints from single-molecule fluorescence resonance energy transfer (smFRET) to describe the superteritary structure of the canonical MAGuK scaffold protein PSD-95. By combining multiple fluorescence techniques, the conformational dynamics of PSD-95 could be characterized across the biologically relevant timescales for protein domain motions. Relying only on a qualitative interpretation of FRET data, we were able to distinguish stable interdomain interactions from freely orienting domains. This revealed that the five domains in PSD-95 partitioned into two independent supramodules: PDZ1-PDZ2 and PDZ3-SH3-GuK. We used our smFRET data for hybrid structural refinement to model the PDZ3-SH3-GuK supramodule and include explicit dye simulations to provide complete characterization of potential uncertainties inherent to quantitative interpretation of FRET as distance. Comparative structural analysis of synaptic MAGuK homologues showed a conservation of this supertertiary structure. Our approach represents a general solution to describing the supertertiary structure of multidomain proteins.

Fig. 1.

Probing domain organization in PSD-95 with analytical size exclusion chromatography. (A) Schematic representation of the domain order in PSD-95. The truncated PSD-95 constructs used in the SEC experiments are depicted in the table. Filled cells indicate the domain composition. For the SEC analysis, each fragment was designated by a letter in line with the associated row. (B) Logarithm of the molecular weight (MW) is plotted against the normalized elution volume (V_e/V_o). Fragments are identified by the letter shown next to each data point. Error bars indicate replicate measurement errors. The black line was calculated using globular protein standards. (C) The difference between apparent molecular weight from SEC and formula weight (ΔMW) is shown for each PSD-95 fragment. Fragments are identified by the letters beneath the plot.

Fig. 2.

Variance of mean FRET distinguishes static and dynamic structures. (A) Variance of mean FRET (VMF) for measurements between PDZ1 and the other domains in PSD-95 (indicated beneath the panel). Measurements between PDZ1 and PDZ2 was measured previously (44). VMF characterizes replicate measurements using different labeling sites to oversample the domain position. VMF between PDZ1 and PDZ2 was significantly higher than measurements to PDZ3 or GuK (* p = 0.046, Brown-Forsythe test for unequal variance) (B) VMF for measurements between GuK and the other domains in PSD-95 (indicated beneath the panel). The VMF between GuK and PDZ3 or SH3 were indistinguishable from each other but significantly higher than measurements to PDZ1 or PDZ2. (** p = 0.048).

Fig. 3.

PDZ3 has a defined position relative to the SH3 and GuK domains. (A) Residues used for labeling in smFRET measurements to PDZ3 are shown as spheres in the carton representation of the GuK domain. Coloring denotes whether these sites showed high FRET (red, K591, Q621 and A640), mid FRET (green, R671 and H702) or low FRET (blue, E572 and S606). (B) Mean FRET for each measurement between PDZ3 and GuK plotted against the grouped labeling positions from panel A. Error bars indicate the standard deviations for each set of measurements. The mean FRET of these groups are statistically different (* p = 0.033, one-way ANOVA). (C) Labeling sites in SH3 (left) and PDZ3 (right) used in interdomain smFRET measurements. Residues used as labeling-sites are shown as spheres. Residues in SH3 are colored according to their FRET dependence in measurements to PDZ3 (cyan; R492 and C445; magenta, T482). (D) Dependence of mean smFRET efficiency on labeling site position for measurements between SH3 and PDZ3. Curves are colored according to the labeling sites as depicted in panel C. The PDZ3 site is denoted beneath the plot.

Fig. 4.

A model for full-length PSD-95 in solution. (A) Best-fit model from rigid body docking using smFRET restraints to position PDZ3 (orange) relative to SH3 (red) and GuK (purple). The SH3-GuK supramodule is shown as surface representation, while PDZ3 is depicted as cartoon representation. Arrow indicates the SH3 HOOK domain. The left and right images are related by a 90° rotation as indicated. (B) Cartoon model for the supertertiary structure of full-length PSD-95. Domain positioning and the average dimensions (indicated in the panel) are in accordance with our smFRET restraints. This represents one snapshot image of a dynamic protein that changes conformation on the submillisecond timescale.

Fig. 5.

Probing PDZ3 dynamics with multiparamater fluorescence detection. PSD-95 was labeled in PDZ3 and (A) GuK (sample #34), (B) SH3 (#44) or (C) PDZ2 (#53) (labeling scheme Table S1). 2D-MFD plots in the upper panel show the FRET indicator, F_D/F_A (ratio of D and A fluorescence) against the donor fluorescence lifetime in the presence of the acceptor (τ_D(A), inset plot, top). The expected curve for static FRET is represented by the dashed line, while that for dynamic FRET is shown in black. The FRET populations identified by PIE are shown in red. The equations for the FRET lines are given in the SI Text, (Eq. S15B). (D) Subensemble lifetime fit for the FRET population confirms the existence of two limiting FRET states with τ₁ = 0.8 ns (34%) and τ₂ = 3.4 ns (66%). (E) Fluorescence cross correlation spectroscopy (FCCS) curve for the FRET bursts confirm the multitime scale dynamics. The data was fit to a diffusion and relaxation model (blue line; see SI Text, Eq. S16). The pure diffusion component is illustrated by the dashed line.

Fig. 6.

Resolving SH3 domain loop configurations with molecular dynamics and smFRET. (A) The root mean squared deviation (RMSD) as a function of time is plotted for the protein backbone relative to the starting structure during an 100 ns all-atom molecular dynamics simulation of the SH3-GuK domain fragment (grey, SH3 domain; black, GuK domain). The SH3 domain showed a rapid reorganization that stabilized after approximately 30 nanoseconds. (B) The root mean squared fluctuation (RMSF) for each C_α atom in the SH3 domain during the molecular dynamics simulation (black line) is plotted along with the RMSF from our ensemble of smFRET-derived models (grey diamonds). Because the core of the SH3 domain was held rigid in our docking calculations, the RMSF can only be calculated for freely moving atoms within the extended loops. The regions containing secondary structural elements (α-helices as cylinders and β-sheets as arrows) are indicated above the panel.

Fig. 7.

Supertertiary structure is conserved across the MAGUK family of scaffold proteins. Each panel shows smFRET histograms for a set of measurements made between homologous positions in PSD-95 (black), SAP97 (dashed) and SAP102 (grey). When shown, the number in each panel indicates the labeling site combination in PSD-95 according to Table S1. Measurements made between (A) PDZ1 and PDZ2, (B) either PDZ1 or PDZ2 and PDZ3 and (C) PDZ3 and the GuK domain. Mean smFRET and peak widths from these experiments are listed in Table S4.