Table 1.
Data collection and refinement statistics.
Figure 1.
Ring structures of SpLsm3, SpLsm4N and SpLsm5/6/7 within crystal lattices.
Structures are viewed from the helix faces of each ring structure. (A) The asymmetric unit of orthorhombic SpLsm3 crystal consists of 14 protein subunits, which are packed into two heptamers coaxially via helix face-helix face region. One SpLsm3 heptamer is shown. (B) The asymmetric unit of monoclinic SpLsm4N crystal contains 24 protein subunits, which are arranged as 8 copies of trimer. One SpLsm4N trimer is shown. (C) The asymmetric unit of tetragonal SpLsm5/6/7 crystal contains one copy of each subunit. Through symmetry operation, a closed hexamer ring structure is generated.
Figure 2.
Overall architectures of SpLsm3, SpLsm4N, SpLsm5, SpLsm6 and SpLsm7.
The monomeric structures of SpLsm3, SpLsm4N, SpLsm5, SpLsm6 and SpLsm7 are shown in cartoon with similar orientations. Each monomer is colored as in Figure 1. The disordered loop 4 region in SpLsm3 and SpLsm7 is shown as dotted lines.
Figure 3.
Sedimentation velocity study of Lsm proteins in solution at 0.75 mg/ml.
The Lsm proteins including SpLsm3, SpLsm5/6/7, SpLsm4N and ScLsm3 were analyzed by sedimentation velocity and fitted based on the c(M) and c(S) size-distribution functions. The corresponding molecular weights obtained from the c(M) size-distribution function for SpLsm3, SpLsm5/6/7, SpLsm4N and ScLsm3 were 75.0 kD, 62.6 kD, 80.3 kD and 11.8 kD, respectively.
Table 2.
Details of sedimentation velocity data analysis.
Table 3.
Oligomeric state of studied Lsm proteins determined from three different concentrations.
Figure 4.
Sequence alignment of Lsm1 to Lsm7 proteins from S. pombe (Sp) and Lsm3 from S. cerevisiae (Sc).
The secondary structural elements of SpLsm3 are shown on top of the sequences.
Figure 5.
Electrostatic potential of Lsm and Sm proteins viewed from the helix and loop faces.
Archaeoglobus fulgidus Sm1 protein (AF-Sm1) (PDB code 1I4K); Pyrococcus abyssi Sm1 (PA-Sm1) (PDB code 1M8V); Homo sapiens Sm complex (HS-Sm) (PDB code 2Y9A); Staphylococcus aureus Hfq (SA-Hfq) (PDB code 1KQ1). The figure was generated with GRASP2.
Figure 6.
Analysis of U15 binding activity of SpLsm2/3, SpLsm3, SpLsm5/6/7 and SpLsm4N.
(A) Sensorgrams of surface plasmon resonance analysis using 5′-end biotin-labeled U15. Fluorescence anisotropy analysis using 5′-end FAM-labeled U15 showed that the fitted Kd value of SpLsm2/3 is 4.0 ± 0.5 µM (B) and the fitted Kd value of SpLsm5/6/7 is 52.5 ± 10.0 µM while no Kd values could be determined for SpLsm3 and SpLsm4N (C).
Figure 7.
Subunit interfaces in SpLsm5/6/7, SpLsm3 and ScLsm3.
Residues involved in interface interaction are shown in stick model. All subunit interfaces are shown in similar orientations. (A) Stereo view of the interface between SpLsm5 and SpLsm6. (B) Stereo view of the interface between SpLsm5 and SpLsm7. (C) Stereo view of the interface between SpLsm6 and SpLsm7. (D) Stereo view of subunit interfaces of SpLsm3. One subunit is colored as in Figure 1 while the other subunit is shown in grey. (E) Stereo view of subunit interfaces of ScLsm3 (PDB code 3BW1). The coloring scheme of the two subunits is as in Figure 7D.