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. 2007 Jan 16;104(3):784-9.
doi: 10.1073/pnas.0610231104. Epub 2007 Jan 10.

Intramolecular interaction in the tail of Acanthamoeba myosin IC between the SH3 domain and a putative pleckstrin homology domain

Kae-Jung Hwang  1 , Fatemeh Mahmoodian, James A Ferretti, Edward D Korn, James M Gruschus
Affiliations

Intramolecular interaction in the tail of Acanthamoeba myosin IC between the SH3 domain and a putative pleckstrin homology domain

Kae-Jung Hwang et al. Proc Natl Acad Sci U S A. .

Abstract

The 466-aa tail of the heavy chain of Acanthamoeba myosin IC (AMIC) comprises an N-terminal 220-residue basic region (BR) followed by a 56-residue Gly/Pro/Ala-rich region (GPA1), a 55-residue Src homology 3 (SH3) domain, and a C-terminal 135-residue Gly/Pro/Ala-rich region (GPA2). Cryo-electron microscopy of AMIC had shown previously that the AMIC tail is folded back on itself, suggesting the possibility of interactions between its N- and C-terminal regions. We now show specific differences between the NMR spectrum of bacterially expressed full-length tail and the sum of the spectra of individually expressed BR and GPA1-SH3-GPA2 (GSG) regions. These results are indicative of interactions between the two subdomains in the full-length tail. From the NMR data, we could assign many of the residues in BR and GSG that are involved in these interactions. By combining homology modeling with the NMR data, we identify a putative pleckstrin homology (PH) domain within BR, and show that the PH domain interacts with the SH3 domain.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
AMIC heavy chain tail. The tail domains are shown schematically with their sequences below. GPA1 and GPA2, glycine/proline/alanine-rich regions; SH3, Src homology 3 domain.
Fig. 2.
Fig. 2.
800 MHz 2D 15N HSQC spectra of the full-length tail of AMIC (A), the BR (B), and the GSG region (C). The BR spectrum is of the sample expressed in 65% D2O. The full-length tail and GSG constructs were expressed in 100% H2O.
Fig. 3.
Fig. 3.
Superposition of the 800 MHz 2D TROSY spectra of the amplitude-adjusted GSG region and full-length tail of the heavy chain of AMIC. (A) The GSG region is in red and the full-length tail in black. (B) Expansion of the region of A showing residues where 1H shift changes occurred between the full-length tail and the GSG region. (C) Expansion of a region where there were no differences between the full-length tail and the GSG region.
Fig. 4.
Fig. 4.
ΔCα–ΔCβ chemical shift index for the basic region construct. Large negative values (less than −4 ppm) are typical of β-strands, extended stretches of large positive values (>4 ppm) are indicative of α-helices, and flexible random coil regions have values closer to zero. Unassigned regions are shown with zero values. Above the graph are β-strands (blue) and helices (pink) from a consensus secondary structure prediction. β-strands and helices contradicted by NMR are shown with dashed rectangles. The strand with the dark blue rectangle is confirmed by NMR. The remaining β-strands lie in unassigned regions. A PH domain predicted by the Phyre threading program is shown at the top.
Fig. 5.
Fig. 5.
Sequence alignments of the putative PH domain of AMIC with PH domains of three other myosins and four nonmyosin proteins. β-strands are in red, helices in green, unassigned regions are indicated by lines above the sequence, and conserved residues are highlighted in yellow. The myosin PH domain secondary structures are predicted, and the nonmyosin secondary structures are experimentally determined.
Fig. 6.
Fig. 6.
Homology modeling of the putative PH domain of the BR of AMIC. (A) The putative PH domain of AMIC (blue) is superposed on the NMR structure of the PH domain of SOS (pink). The seven β-strands are numbered. The PH domain of AMIC BR lacks the C-terminal helix and contains a six residue insert between strands one and two (bottom) not seen in the SOS PH domain. Both PH domains have a mobile loop between strands three and four (left). (B) Electrostatic potential energy surface for the putative PH domain of AMIC BR, shown in the same orientation as in A. The polarization between the mildly negative C-terminal area of the domain (right) and the very positive opposite side and adjoining mobile loop region (left) is typical of PH domains.
Fig. 7.
Fig. 7.
ΔCα–ΔCβ chemical shift index for the GSG construct. For significance of values, see legend to Fig. 3. Predicted β-strands confirmed by NMR (dark blue rectangles) are shown above the graph. The position of the SH3 domain predicted by BLAST and confirmed by NMR is shown at the top.
Fig. 8.
Fig. 8.
Comparison of the backbone amide chemical shift differences between the 15N HSQC spectra of full-length AMIC tail and the BR and GSG regions. Unassigned regions and proline residues are shown with zero values. (A) GSG construct: The accuracy of the chemical shift differences is <0.01 ppm. The dominant differences occur in the SH3 domain. (B) BR construct: The accuracy of the chemical shift differences is ≈0.01 ppm; the largest differences occur in the putative PH domain.
Fig. 9.
Fig. 9.
Surface view of the SH3 domain of AMIC. The region perturbed by interactions with residues in the basic region is shown in yellow, the region that is expected to be the binding site for proline-rich ligands is in green, and the overlap between these two regions is in white. The position of a hypothetical ligand (purple ribbon) is modeled by superposition of the Fyn proto-oncogen tyrosine kinase SH3 domain/ligand complex (Protein Data Bank ID Code 1A0N).
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