Structural Description and Functional Validation of the Knob and Post ActinBinding Sites of the FH2 Domain
In the crystal, each bridge contacts two actin monomers through two distinct sites on
opposite ends of the structure (Fig. 1b). One site is in the knob subdomain and contacts
the barbed end of one actin monomer, burying a total of 1120 Å2 of solvent accessible
surface (Fig. S2a). Binding of the knob is anchored by insertion of the D helix of the
FH2 domain into the cleft between actin subdomains 1 and 3. The highly conserved FH2
residue Ile1431 is located at the center of the interface, where it contacts a hydrophobic
pocket in the actin cleft formed by residues Tyr143, Gly146, Thr148, Ile345 and Leu349.
Adjacent sidechains on the exposed face of D, including Asp1424, Gln1427, Gln1428,
Gly1430, Asn1432, His1434 and Ser1437, make polar contacts to surrounding residues
on subdomains 1 and 3. These interactions resemble those observed for gelsolin, vitamin
D binding protein and cibulot, which all insert the hydrophobic face of an  helix into the
cleft between the two actin lobes1. Additional contacts are made by residues Ser1460 and
Glu1463 on FH2 helix G to actin residues in loop 143-148 and strand 328-331 of
subdomain 3. Finally, Lys1639 also makes a polar interaction with Ser323 and the Cterminus of helix 308-321 in actin subdomain 3.
Helix C (residues 1410-1416) in the inter-bridge linker also contacts actin at the
knob site. However, electron density is poor throughout this region, and sidechain
density is observed only for Asp1414. Residues in C are not conserved, although they
are charged in most formins. The rearrangement to reverse the domain swap in the
crystal requires a reorientation of C, that eliminates its contacts to one of the two actins
held by the bridge (actin 3, Fig. 1c). Thus, in the FH2 complex with filaments, the knob
site interaction may be stronger to one actin (actin 2, green bridge) than the other (actin 3,
blue bridge).
These energetics may contribute to the proposed FH2 domain
configurational equilibrium described in the main text.
The second FH2-actin interface involves a composite surface formed by elements
from both the lasso and post regions of the FH2 domain, which we will refer to as the
post site (Figs. 1b, S2b). This surface binds subdomain 1 on the side of a second actin
monomer, which is related to the knob-bound actin through pseudo-short-pitch symmetry
(21 screw axis of the crystal). Interactions at the post site bury a total of 940 Å2 surface
area, and are mediated largely by the highly polar, basic surface created by sidechains
from helix N (Gln1595, Arg1596, Lys1601) and the M-N loop of the post
(Lys1584), the loop 1359-1362 of the lasso (Lys1359, Gln1360, His1362), and Nterminal residues in the inter-bridge loop (Glu1403, Lys1405, Ser1406).
These lie
opposite an array of acidic and polar residues in actin emanating from subdomain 1
(Asp80, Glu83, Glu117, Lys118, Gln121, Glu125, Lys359, Tyr362, Asp363, Glu364).
Thus, the post site interaction appears to be stabilized largely by polar contacts and
charge complementarity.
To examine the functional importance of the knob and post site interactions, we
tested a battery of FH2 domain mutants in an actin filament assembly assay. Mutation to
alanine of Ile1431, His1434 or Lys1639 in the knob site, or Lys1359, Gln1360, His1362,
Lys1584 or Lys1601 in the post site impairs the ability of the FH2 domain to nucleate
new actin filaments (Fig. S2c, d). The Ile1431 and Lys1601 mutations exhibit the most
severe defects and completely abolish the activity of the FH2 domain in this assay, as
observed previously 2. We also mutated five residues at the only other site of FH2-actin
contact in the crystal, between FH2 helices R and T and actin subdomain 4. This contact
does not involve conserved residues, and none of these mutations affected actin assembly
activity (Fig. S2e). Thus, the knob and post sites are uniquely sensitive to mutation of
interface residues observed in our crystal structure.
To further characterize FH2-actin interactions outside of the crystal, we also
examined binding of the FH2 domain of mDia1 to TMR-actin using NMR spectroscopy
(Fig. S2f). In a sample selectively labeled with 1H-13C at the methyl groups of Val, Leu
and Ile ( only) in an otherwise fully deuterated background, we observed all 11 of the
expected Ile  resonances in 1H-13C TROSY HMQC spectra3. Addition of 25 mole %
TMR-actin to this sample caused substantial broadening of only 1 resonance, which was
subsequently identified by mutagenesis as the Ile845  methyl group (corresponding to
Ile1431 in Bni1p).
Such broadening is likely to be most severe for direct contact
residues, due to a combination of diminished TROSY effect from the proton bath in the
unlabeled actin and chemical exchange. These data suggest that Ile845 in the mDia1
FH2 domain, and by analogy Ile1431 in the Bni1p FH2 domain, is part of the TMR-actin
interface in solution.
Finally, we note that all surface exposed residues that are conserved in >50% of the
54 FH2 domain sequences in the Pfam database4 are located in either the knob or post
interfaces. Together, these data strongly support the functional relevance of both the
knob and post sites of FH2-actin contact in the crystal.
References
1.
Dominguez, R. Actin-binding proteins - a unifying hypothesis. Trends Biochem
Sci 29, 572-8 (2004).
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4.
Xu, Y. et al. Crystal structures of a Formin Homology-2 domain reveal a tethered
dimer architecture. Cell 116, 711-23 (2004).
Tugarinov, V., Hwang, P. M., Ollerenshaw, J. E. & Kay, L. E. Cross-correlated
relaxation enhanced 1H-13C NMR spectroscopy of methyl groups in very high
molecular weight proteins and protein complexes. J Am Chem Soc 125, 10420-8
(2003).
Bateman, A. et al. The Pfam protein families database. Nucleic Acids Res. 30,
276-280 (2002).