1. Recognition of eIF4G by Rotavirus NSP3 Reveals a Basis for mRNA Circularization 
Caroline M. Groft, Stephen K. Burley 
Molecular Cell 
Volume 9, Issue 6, Pages (June 2002)
DOI: /S (02)

2. Figure 1 Structure of the NSP3-C/eIF4G Heterotetramer
(A) BOBSCRIPT (Esnouf, 1999) drawing showing the NSP3-C homodimer, with each monomer in gold or blue. NSP3-C α helices are labeled H1–H3, and the C-terminal β strand (S1) forms a sheet with the N terminus of the eIF4G fragment. Two eIF4G molecules are shown in red and green. eIF4G β strands are labeled S1 and S2 and the α helix is labeled H1. The N- and C termini of the monomers and the peptides are also indicated.
(B) BOBSCRIPT drawing of (A) turned 90° toward the reader.
(C) BOBSCRIPT drawing of the Max homodimer bound to DNA (Ferre-D'Amare et al., 1993) with each monomer in gold or blue and the N- and C termini of the monomers labeled. The DNA backbones are drawn as red and green ribbons.
Molecular Cell 2002 9, DOI: ( /S (02) )

3. Figure 2 Sequence Alignments of Group A NSP3 eIF4G Binding Domains
(A) Sequence alignment of Group A NSP3 eIF4G binding domains. Secondary structural elements were obtained from the X-ray structure and are identical for both NSP3 monomers. Circles denote disordered residues. The last three residues are disordered in the NSP3 blue monomer (gray circles) but could be modeled in the gold monomer (black “b”). Sequence similarity is encoded by a yellow→green color gradient (40%–100% identity). Functional classifications: red “P” or teal “P,” residues that contribute to peptide binding by either monomer to a single peptide binding site; purple “D,” residues that contribute to dimerization.
(B) Sequence alignment of eIF4G and eIF4B peptides. Secondary structural elements were obtained from the X-ray structure and are the same for both peptides. Functional classifications: $, intrapeptide contacts with NSP3-C (red→gold monomer or green→blue monomer); *, contacts with NSP3-C (red→blue monomer or green→gold monomer).
Molecular Cell 2002 9, DOI: ( /S (02) )

4. Figure 3 NSP3-C Recognition of eIF4G
(A) Hydrophobic interactions between the red eIF4G fragment and the NSP3-C homodimer. The gold monomer is drawn in full with a semitransparent blue monomer. Aromatic side chains are shown with blue planes.
(B) Polar interactions between the red eIF4G fragment and the NSP3-C homodimer.
Molecular Cell 2002 9, DOI: ( /S (02) )

5. Figure 4 Surface Properties of NSP3-C
(A) GRASP (Nicholls et al., 1991) surface representation of NSP3-C color coded yellow for underlying conserved residues.
(B) GRASP (Nicholls et al., 1991) representation of the chemical properties of the solvent-accessible surface of NSP3-C calculated using a water probe radius = 1.4 Å. The surface electrostatic potential is color coded red and blue, representing electrostatic potentials <−10 to >+10 kBT, where kB is the Boltzmann constant and T is the temperature (Gilson et al., 1988).
The eIF4G fragment is shown as a green α-carbon backbone.
Molecular Cell 2002 9, DOI: ( /S (02) )

6. Figure 5 Biophysical Studies of eIF4G Binding by NSP3-C and PABP
(A) Left: Sample titration curve of NSP3-C(206–315) with wild-type eIF4GI(132–160). Right: Sample titration curve of wild-type PABP(1–190) with wild-type eIF4GI(132–160).
(B) GST pull-down assay to assess biochemical activity of the isolated NSP3-C domain. “Input” lanes show sizes of the different fragments used in the assay. “GST-eIF4G(132–160),” GST-eIF4G protein was incubated with resin only; “GST-eIF4G/NSP3-C,” GST-eIF4G protein was incubated with non-tagged NSP3-C protein before resin addition; “GST-PCBP/NSP3-C,” a fragment of an unrelated protein, GST-PCBP(287–365), was incubated with non-tagged NSP3-C prior to resin addition. The black arrow indicates the position of NSP3-C.
(C) Summary of PABP(1–190)/poly(A)11 RNA and PABP(1–190)/eIF4G(132–160) equilibrium dissociation constants obtained from nitrocellulose filter binding assays and ITC, respectively. N.D. denotes dissociation constants that could not be determined due to lack of sufficient heat evolution, which reflect Kd>>300 μM.
(D) Sequence of eIF4GI used for ITC experiments. Underlined residues (I140, I142, I152, and I155) were individually changed to Ala, giving mutant eIF4G fragments that showed no evidence of binding to wild-type PABP(1–190).
Molecular Cell 2002 9, DOI: ( /S (02) )

7. Figure 6 Comparison of NSP3-C and PABP-RRM2 Surfaces
(A) GRASP (Nicholls et al., 1991) surface representation of the first two RRMs of PABP. Sites of mutations deleterious to eIF4G binding are shown in magenta, and the hydrophobic groove is indicated with blue-gray. Mutations that do not affect eIF4G binding are shown in teal.
(B) RIBBONS drawing of RRM2 of PABP showing RNA and select side chains in gold and purple atomic stick figures, respectively.
(C) RIBBONS drawing of the NSP3-C peptide binding pocket, with select hydrophobic side chains shown as purple stick figures.
Molecular Cell 2002 9, DOI: ( /S (02) )