(Received for publication, September 20, 1995; and in revised form, October 20, 1995)
From the
The Escherichia coli protein GroES is a co-chaperonin that is able to assist GroEL in the refolding of proteins. GroES is a heptamer of seven identical subunits. Recent work has focused on the structural aspects of GroES. We have investigated the role of the C-terminal portion of GroES on its oligomerization. Limited proteolysis of GroES by carboxypeptidase Y gives a product in which the C-terminal 7 amino acid residues have been removed. Sedimentation velocity analysis reveals that the truncated form of GroES is unable to reassemble. The results presented here implicate the C-terminal sequence in intermonomer actions within the GroES oligomer. In addition, this work provides experimental verification of predictions implied in the recent x-ray determination of the GroES structure (Hunt, J. F., Weaver, A. J., Landry, S. J., Gierasch, L. M., and Deisenhofer, J. Nature, in press).
The chaperonins are proteins that are essential for cell growth under normal conditions(1) . Among their various functions, chaperonins have been shown to assist in the folding of many proteins. Widely studied representatives of the chaperonin family are the GroES and GroEL proteins of Escherichia coli(2, 3) . Recent models for chaperonin action have the larger of the two proteins, GroEL, binding partially folded protein intermediates(4) . GroES participates in the cycle by modulating the ATPase activity of GroEL(5, 6, 7) . In addition, the release of partially folded polypeptides has been proposed to be modulated by GroES(8, 9, 10) , possibly through the interaction of a mobile loop on GroES with GroEL(11) .
Structurally, GroES is a heptamer composed of seven identical 10-kDa
subunits(6) . The subunits are organized into a single ring
structure. An x-ray crystal structure of the oligomer has been recently
solved and shows the core of the protein to be organized as a
topologically irregular -barrel. (
)A recent study has
shown that there exists a dynamic equilibrium between monomeric and
oligomeric GroES(12) . In that report, GroES was shown to
dissociate with a half time of a few minutes and a dissociation
constant of 1
10
M
(12) . Those results indicate that, as
with GroEL(13) , monomeric GroES may play an important
functional role in the mechanism of chaperonin action.
We have
investigated the role of the C-terminal region of GroES in the
oligomerization of the protein. The C-terminal sequence of GroES is
resistant to proteolysis by carboxypeptidase Y in the absence of
structural perturbants. However, in the presence of moderate
concentrations of urea, a portion of the C-terminal sequence of GroES
becomes susceptible to proteolysis. Under conditions that have been
previously shown to allow for reassembly of urea denatured GroES, ()the truncated species is no longer able to oligomerize.
Amino acid analysis reveals that the C-terminal 7 amino acid residues
are released from GroES, giving the truncated species. Structurally,
these residues appear to be important for the integrity of the monomer
interface.
Amino acid analysis was performed using a Beckman 7300 analyzer using the ninhydrin reaction after the amino acids had been separated on a 20-cm high resolution ion exchange column. Detection of the derivatized amino acids was at 570 and 440 nm. The retention time and peak heights of the samples were compared with known standards to determine amino acid identity and quantity. The results from the amino acid analysis were compared to the published sequence of GroES to determine the site of truncation(18) .
Figure 1: Proteolysis of GroES by carboxypeptidase Y. GroES was subjected to limited proteolysis in the presence of urea as described under ``Experimental Procedures.'' Samples were incubated in the concentrations of urea denoted in the header of each set of lanes (0M, 3M, and 4M). ES denotes a native GroES standard (panel B only). After proteolysis an aliquot of the product was diluted into an equal volume of 9 M urea (lanes marked U) or into an equal volume of buffer (lanes marked B). Native GroES is denoted by N, and truncated GroES is marked by T. Panel A, SDS-polyacrylamide gel electrophoresis. Panel B, nondenaturing electrophoresis.
The proteolytic
product also demonstrates an altered mobility during native gel
electrophoresis (Fig. 1B, lanes 3M and 4M). Conditions were chosen from previous
studies that would allow for reassociation of oligomers by
dilution into an equal volume of buffer followed by electrophoresis (lanes denoted by B). Likewise, dilution of the
sample into an equal volume of 9 M urea (lanes marked U) would allow for the dissociation of oligomeric GroES. A
comparison in Fig. 1B of lane ES with lane
U at 0 M urea shows that electrophoresis allows for the
reassembly of samples that were originally at concentrations as high as
4.5 M urea. The digestion product has an altered mobility
irrespective of the dilution conditions prior to electrophoresis.
Analysis of the amino acids released from the limited proteolysis of GroES give the following ratios of amino acids: A:I:E:V:L, 2:2:1:1:1. When matched with the published sequence of GroES(18) , this sequence corresponds to the 7 C-terminal amino acids of GroES (highlighted in bold): . . . EVLIMS.E.SDILAIVEA.
This sequence corresponds to the predicted C-terminal strand in the GroES structure(20, 21) . One of these reports implicates this C-terminal strand in intermonomer interactions(21) .
Figure 2:
Sedimentation velocity analysis of
truncated GroES. GroES was proteolyzed with carboxypeptidase Y and
subjected to reassembly as described under ``Experimental
Procedures.'' Data were collected and analyzed as described under
``Experimental Procedures.'' Analysis is presented as the
integral distribution of S values for GroES. The y axis
indicates the fraction of material with s values less than or equal
to the values given on the x axis. A single, pure component
displaying ideal behavior would be represented by a vertical line that intercepts the x axis at the appropriate s
value.
Several recent reports have focused on the
structural characteristic of
GroES(20, 21) . It is believed that the
oligomeric structure of GroES serves to coordinate the hydrolysis of
ATP(22, 23, 24) . It has been reported
recently that GroES dissociates at relatively low
concentrations(12) . The dissociation of GroES suggests that
the monomeric species may play a role in the chaperonin mechanism.
The present work emphasizes the importance of the C-terminal
sequence of GroES in the oligomerization of the protein. When these
results are analyzed in the context of the recently solved x-ray
crystal structure, the reasons become clear. The C-terminal
7 residues form the last
-strand. This
-strand makes the
primary contacts with the N terminus of a neighboring monomer forming
the interface between monomers. This interface is rich in flexible side
chains, and the interface is described as having structural plasticity.
In summary, the production of monomeric GroES by limited proteolysis
provides a new avenue for the study of GroES function.