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.2017 Jun 1;34(6):1463-1478.
doi: 10.1093/molbev/msx094.

Structural History of Human SRGAP2 Proteins

Affiliations

Structural History of Human SRGAP2 Proteins

Michael Sporny et al. Mol Biol Evol..

Abstract

In the development of the human brain, human-specific genes are considered to play key roles, conferring its unique advantages and vulnerabilities. At the time of Homo lineage divergence from Australopithecus, SRGAP2C gradually emerged through a process of serial duplications and mutagenesis from ancestral SRGAP2A (3.4-2.4 Ma). Remarkably, ectopic expression of SRGAP2C endows cultured mouse brain cells, with human-like characteristics, specifically, increased dendritic spine length and density. To understand the molecular mechanisms underlying this change in neuronal morphology, we determined the structure of SRGAP2A and studied the interplay between SRGAP2A and SRGAP2C. We found that: 1) SRGAP2A homo-dimerizes through a large interface that includes an F-BAR domain, a newly identified F-BAR extension (Fx), and RhoGAP-SH3 domains. 2) SRGAP2A has an unusual inverse geometry, enabling associations with lamellipodia and dendritic spine heads in vivo, and scaffolding of membrane protrusions in cell culture. 3) As a result of the initial partial duplication event (∼3.4 Ma), SRGAP2C carries a defective Fx-domain that severely compromises its solubility and membrane-scaffolding ability. Consistently, SRGAP2A:SRAGP2C hetero-dimers form, but are insoluble, inhibiting SRGAP2A activity. 4) Inactivation of SRGAP2A is sensitive to the level of hetero-dimerization with SRGAP2C. 5) The primal form of SRGAP2C (P-SRGAP2C, existing between ∼3.4 and 2.4 Ma) is less effective in hetero-dimerizing with SRGAP2A than the modern SRGAP2C, which carries several substitutions (from ∼2.4 Ma). Thus, the genetic mutagenesis phase contributed to modulation of SRGAP2A's inhibition of neuronal expansion, by introducing and improving the formation of inactive SRGAP2A:SRGAP2C hetero-dimers, indicating a stepwise involvement of SRGAP2C in human evolutionary history.

Keywords: F-BAR domain; SRGAP2; X-ray crystallography; human evolution; structural biology.

© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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Figures

F<sc>ig</sc>. 1
Fig. 1
Evolutionary history and domain organization of SRGAP2 proteins. (A) Evolutionary history diagram detailing the duplication (Dup.) and mutagenesis (Mut.) events in humanSRGAP2 genes and their current status in modern-day humans. A genetic approximate timeline and parallel stone tools technology archaeological dating (Harmand et al. 2015) shows correlation between the first and second mutagenesis events with the first and second generations of stone tools, ∼3.3 and ∼2.4 Ma, respectively. The missing carboxy-segment of the Fx in all the duplicated copies is indicated as a zig-zag tear followed by a seven-residue (VRECYGF) carboxy-terminal addition. Also indicated and detailed are the nonsynonymous mutations in the modern duplicated proteins. The primal forms ofSRGAP2B andSRGAP2C, which existed prior to the accumulation of mutations are named with the prefix “P-.” (B) Human SRGAP2A, SRGAP2C, and deletion mutants used in this study. In the scheme, the extended F-BAR (F-BARx) is subdivided into the F-BAR and the newly identified Fx domains. The RhoGAP and SH3 domains are also indicated and appear as yellow colored shapes. The five arginine alterations in the F-BAR domain of SRGAP2C are indicated by ticks.
F<sc>ig</sc>. 2
Fig. 2
SRGAP2A structure and dimerization. (A) Domains of SRGAP2A and focus on F-BARx. The F-BARx helices and encoding exons are drawn in scale and designated by their numbers. The carboxy-half of helix 6, encoded by exon 10, which is missing in SRGAP2C, is depicted in black. (B) Ribbon diagrams and cartoons of the dimeric SRGAP2A F-BARx crystal structure, in which the two identical protomers are colored in yellow and in cyan. Labels indicating structural elements of the cyan protomer are underscored. The large ribbon diagram represents a view facing the N-surface, and delineates the 6-helix core, arms, and tip regions. (C) The RhoGAP-SH3 domains bind directly to the F-BAR domain. Microscale thermophoresis (MST) titration curves and apparentKD values for RhoGAP-SH3 binding to the F-BAR or the F-BARx that were fluorescently labeled. Measured thermophoresis values (symbols) fitted to a one-to-one binding model (solid lines). (D) 3D domain organization of SRGAP2A homo-dimers (left and center) and SRGAP2A:SRGAP2C hetero-dimers (right). SAXS 3D volume of full-length SRGAP4 (first shown in Guez-Haddad et al. [2015]) is represented as a transparent envelope into which the crystal structures of the F-BARx and the RhoGAP-SH3 domains were docked after being reduced to a 25 Å resolution 3D volumes by CHIMERA (Yang et al. 2012). SRGAP2C is colored in red and is missing the RhoGAP-SH3 domains as well as part of the Fx. Therefore, it forms an asymmetric hetero-dimer with SRGAP2A that has lesser dimeric interface than the SRGAP2A homo-dimer.
F<sc>ig</sc>. 3
Fig. 3
An inverse F-BAR curvature in SRGAP2A. (A) The crystal structures of the SRGAP2A F-BARx homodimer (yellow and cyan) and the FBP17 F-BAR (PDB code 2EFL, both protomers colored in gray) are superimposed. While the six-helix core segments are well aligned, the arms of the SRGAP2A F-BARx point in opposite directions to those of the FBP17, thereby generating a convex N-surface. This view of the superimposed α2-α3 arms shows a ∼30° inversion in the SRGAP2A F-BARx arm curvature. Colored in red is the 25 aa insertion segment in helix α2 that imposes the curvature inversion. Residue Val100 of FBP-17 is colored in green. (B) Maximum intensity projections of COS-7 cells transfected with GFP and GFP-fusion of the SRGAP2A F-BARx. Upper panel is a magnification of an area boxed in the image. Note that F-BARx-expressing cells are marked by high frequencies of protrusions from the plasma membrane. Bars, 10 µm. Quantitative determinations of protrusion frequencies at the perimeter of cells transfected as indicated. Data are mean + SEM. Statistical significances were tested using one-way ANOVA with Kruskal–Wallis and Dunn’s post-tests. *P < 0.05; ***P < 0.001. (C) Maximum intensity projections of COS-7 cells transfected with GFP-fusion of the FBP17 F-BAR domain shows formation of membrane invaginations, a typical phenotype of canonical F-BAR domains. (D) While canonical F-BAR domains (e.g., FBP-17) that have a concave membrane binding surfaces associate with membrane invaginations and participate in endocytic processes, SRGAP proteins have an inverse, convex membrane binding surface that allows them to associate with membrane protrusions, for example, lamellipodia and dendritic spine heads.
F<sc>ig</sc>. 4
Fig. 4
SRGAP2A-membrane electrostatic interactions. (A) Electrostatic surface potential of F-BARx homodimer, projected onto its molecular surface using PyMOL (Schrödinger LLC). Note the contrast between the predominantly electropositive N-surface and the electronegative opposite surface. Key residues are indicated, and residues from one protomer are underlined while those of the second protomer are not. Fx regions are circled. (B) SRGAP2A F-BARx association with membranes is mediated by electrostatic interactions, as SRGAP2A F-BARx cosedimentation with liposomes is reduced by increasing concentrations of phosphate. Moreover, a pre-bound F-BARx is released by addition of phosphate (right panel). Supernatant (S) and pellet (P) fractions are indicated. (C) Gel densitometry quantification of liposome-bound fractions from cosedimentation experiments of SRGAP2A and SRGAP2C proteins, incubated with increasing concentrations of 400 nm sized liposomes composed from porcine whole brain extract lipids. The error bars indicate SEM; see supplementary figure S6, Supplementary Material online for SDS-PAGE Coomassie stained gels. (D) COS-7 cells expressing the F-BARx-R5E mutant show smooth surfaces, when compared with the F-BARx-WT in figure 3B. Quantitative protrusion analysis as in figure 3B.
F<sc>ig</sc>. 5
Fig. 5
SRGAP2C is insoluble due to Fx truncation. (A) GFP fusion of SRGAP2C expressed in COS-7 cells does not form membrane protrusions when compared with F-BARx, and shows a granulated cytosolic distribution. (B) When expressed alone in Sf9 cells, SRGAP2A is found exclusively in the soluble fraction. Coomassie-stained SDS-PAGE (left) and anti-SRGAP2A (abcam, ab124958 directed against the carboxy-tail of the protein that is not present in the SRGAP2C) western-blot analysis. SRGAP2A migrates next to the 135kDa marker. His-tagged SRGAP2A was expressed in Sf9 cells using the Bac-to-Bac system. P3 infected Sf9 cells (from one 2-l roller bottle, Greiner Bio-One) were harvested 72 h postinfection and lyzed in phosphate lysis buffer. The cell extract was centrifuged and the soluble fraction loaded onto a Ni-chelate column. Imidazole gradient was applied and the His-tagged SRGAP2A eluted at 100 mM imidazole. (C andD) His-tagged SRGAP2C (C) and His-tagged P-SRGAP2C (D) are found exclusively in the insoluble fraction. Both proteins were expressed as in (B) and analyzed using Coomassie-stained SDS-PAGE and anti-His western-blot analysis. Both SRGAP2C and P-SRGAP2C migrates below the 63 kDa marker. (E) Cartoon, ribbon, and electron density map representation of the missing-in-SRGAP2C exon 10 structure. Exon 10 encodes for the carboxy-half of helix α6 that mediates the Fx interactions with the F-BAR α2–α3 arm and is thereby essential for the Fx fold integrity. In its absence, the amino-half of helix α6 and the entire α5 helix loses their hold on the F-BAR arm and the structural integrity of the Fx. Fx misfolding due to the missing exon 10 readily explains SRGAP2C and P-SRGAP2C insolubility. A simulated annealing composite omit electron density map contoured to 1.3σ, highlighting the close contacts of helix 6 with helix 5 of the Fx, and with the F-BAR portion of the reciprocal F-BAR protomer.
F<sc>ig</sc>. 6
Fig. 6
SRGAP2A:SRGAP2C hetero-dimerization. (A–C) Coomassie-stained SDS-PAGE (A) and western-blot analysis (B) show that, in sharp contrast to SRGAP2A solubility (fig. 5B), large amounts of (nontagged) SRGAP2A are in the insoluble fraction when coexpressed with either His-tagged SRGAP2C or His-tagged P-SRGAP2C in Sf9 cells. Important to note that the nonHis-tagged SRGAP2A binds to the Ni-chelate column and elutes at 50 mM imidazole. SRGAP2C and P-SRGAP2C remain insoluble under coexpression conditions. (C) Gel densitometry quantification of SRGAP2A soluble fraction (calculated from [A] and fig. 5B), as the ratio between the total amount of protein in the Ni-column elution and the insoluble fraction. The error bars indicate SEM. (D–F) Coomassie stained SDS-PAGE of superose6 10/300 elution profiles, and SEC-MALS analysis of SRGAP2A (residues 1–799, F-BARx-RhoGAP-SH3) alone (D), coexpressed with F-BARA (SRGAP2A residues 1–355) (E), or coexpressed with F-BARC (SRGAP2C residues 1–355) (F). Note that while the SRGAP2A:F-BARA complex dissociates into a homo- and hetero-dimers mixture under gel-filtration conditions, the SRGAP2A:F-BARC hetero-dimers are remarkably stable. An elaborate description of the SRGAP2A:F-BARC and SRGAP2A:F-BARA hetero-dimers production is presented in supplementary figure S7, Supplementary Material online. (G) Coomassie stained SDS-PAGE of superose6 10/300 elution profiles, and SEC-MALS analysis of F-BARC.
F<sc>ig</sc>. 7
Fig. 7
The SRGAP2C R108W substitution inflicts structural instability. F-BARA (A) and F-BARC (B) were expressed as -TEV-His-Trx fusions inE. coli. The proteins were eluted by an imidazole gradient from Ni-NTA column, digested by TEV protease, and further purified by a superdex200 20/60 gel filtration column. (C) The purified F-BARA and F-BARC, as well as each one of the SRGAP2C point mutants introduced to the F-BARA template (which were expressed and purified like F-BARA and F-BARC) were analyzed by circular dichroism (CD) spectroscopy in 10 °C. All seven F-BAR constructs show similar CD spectra, with a predominantly alpha helical secondary structure content. (D) Temperature gradient CD analysis (average of three runs) shows a reduced thermal stability and structural cooperativity of F-BARC and the R108W mutant in comparison to F-BARA. (E) Calculated denaturation temperatures (Td) and denaturation slopes are presented for each F-BAR construct. (F) Zoom in on the R108 region and interactions.
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