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Interallelic complementation and allostery
J. Mol. Bid. (1968) 37,239-242
Interallelic Complementation and Allostery The defects responsible for loss of activity in mutant forms of enzymes need not always involve the active sites themselves and could involve parts of the peptide oh&n associated either with, or with the conditioning of, conformational change. The point of this letter is to show that if this is so, then extensive complement&ion might result without the need for homologous correction. It is fairly generally accepted that interallelic complementetion is something which results from the interaotion of subunits in multimeria proteins. (For recent reviews e.eeFinaham, 1966, and Gillie, 1966.) The most plausible theories of the effect-those of Kapuler & Bernstein (1963) and of Crick & Orgel (1964)-hsve involved homologous oorrection. It has been assumed in other words that the close juxtaposition of a defeotive region in one monomer with a correct homologous region of another monomer can result in the correction of the defect. As pointed out by Crick & Orgel the models of protein structure invoked by Kepuler b Bernstein are implausible. Crick & Orgel themselves suggest that multimerio proteins are likely to be symmetrical and in particular that they are likely to involve rotation axes. They also suggest that correction is most likely to take place in the vicinity of the rotation axes. As Crick & Orgel point out, their theory does lead to the general type of relationship observed in oomplementation. In what follows it is eccepted that multimeric proteins are likely to be symmetrical in the way that C&k & Orgel suggest. Crick t Orgel dismiss 8s a special case complementation between monomers where the active site is made up of parts of two or more subunits. (This special c&se is shown diagrammatically in Figure 1). They consider that this may be oorrect in some cases but that it can hardly provide a basis for a general theory of the approximate linearity of complement&ion maps spreading over considerable lengths. Thus with the arrangement of Figure 1 all the complementing mutants would fall into two groups, (see also Fincham, 1966). It seems, however, that the mechanism of this speoial ease, simple juxtaposition of parts of active sites, unlike that of homologous oorreotion, is one which simply must work. It therefore seemed worth asking if there are any ways in which it might be extended. Another reason for looking for some general explanation of complement&ion in addition to the homologous correction one of Crick & Orgel, is that complement&ion maps are often basically simple. Also, (see for example Gillie, 1966) in many cases a high proportion of mutants show complement&ion, whioh is diffioult to account for on the basis of homologous correction in the vicinity of a.xes of symmetry. It seems possible to extend the simple juxtaposition theory (that of Fig. 1) by considering that an enzyme might be rendered inaative by damage to sny one of several sites, possibly its allosterio binding sites. The simplest ease of this kind involves a dimerio allosteric enzyme, eeoh monomer of which has two sites, 8 and A (possibly substrtlte and activator binding sites) each complete in itself. If it is assumed that for activity all that the dimer requires is one aotive A site and one adive S site, then this 239
S. McGAVIN
240
FIG. 1. Diagram of the simple “special case”. If both A’s or both B’s are damaged the dimer is inactive. If an A in one monomer and a B in the other are damaged the dimer is active since it has one undamaged AB site.
(a)
’ A,(b)
I I ,f
I -- A
s
S'
-I-
'
S'd
SA AS'
-+----
A'
S
I II ---v--I I
I I
FIG. 2. (a) Diagram of an enzyme structure consisting of two identical peptide chains related by a twofold axis of rotation. The structure has two sites (SS’ and AA’) essential to its activity. In active site SS’, S is considered to come from one subunit and S’ from the other and in AA’, A is considered to come from one subunit and A’ from the other. SS’ and AA’ might be substrate binding and activitor binding sites. The structure is shown as a cylindrical development. The relationship of S and A at the left of the diagram to S’ and A’ at the right is the same as the relationship of S and A to S’ and A’ at the centre. (b) Complementation map resulting from the model of Fig. 2(a). The map can be folded into a circle. TABLE
1
Compkmentation properties of the model shown in Pig. 2(a)
S S’ A A’ SS’ AA’ SA SA S’A S’A’
S
S’
A
A
SS’
AA’
0 + + + 0 0 0 0 + +
+ 0 + + 0 0 + + 0 0
+
+ + + 0 0 0 + 0 + 0
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
SA
SA’
S’A + 0 0 + 0 0 0 + 0 0
S’A’ + 0 + 0 0 0 i0 0 0
S, S’, A and A’ are groups of mutants which make half sites S, S’, A and A’ inactive. SS’, AA’, etc. are groups of mutants which make pairs of half sites S and S’, A and A’, SA etc. within one monomer inactive. + indicates that groups of mutants could complement. 0 indicates that groups of mutants would not complement.
LETTERS
TO
THE
EDITOR
241
results in complement&ion properties similar to those of the “special case” discussed above (Fig. 1). If, however, each site is made up of parts of the two subunits, then the number of complementing groups can increase considerably. This is illustrated by means of a model shown in Figure 2(a) and in Table 1. There are now two kinds of groups of complementing mutants, one kind affecting only one-half site, the other affecting two different half-sites within a monomer. Mutations affecting three half sites would not complement in this case. The resulting complement&ion map involving four units and eight groups is shown in Figure 2(b). The number of complementing groups can be further increased and the map made more complex if damage to a third site, again consisting of two half sites, could render the enzyme inactive. In this case some mutations affecting three half sites will complement. One such system, for example, can result in a map involving six units and twenty-six complementing groups, eighteen of which, those most likely to occur, can be included in a linear map. The pattern of complementation could be simplified in either of these cases if some of the specific sites were complete within monomers. It could be made more complex if sites are contributed to by more than two monomers, or if more than three sites are essential to activity. So far defects associated with specific binding sites have been considered as examples. Allosteric enzymes, however, are presumably structures which are delicately balanced between two or more states (see for example Monod, Wyman C%Changeux, 1965), damage to many different sites within or on an allosteric protein could perhaps interfere with its moving from one state to another or could lock it in one state. It seems possible therefore that mutations not associated directly with any of the specific binding sites might be able to tip the balance one way or the other and that this loss of balance might be corrected by another mutant thus giving rise to further complement,ing groups. If mutations could cause the loss of, or the correction of a rather delicate balance then it seems that “bad correcting bad” might be involved. An important point here which bears particularly on mutations affecting conformational changes, rather than the active sites themselves, is that in a symmetrical mutant homomultimer the damage is also symmetrical, in the corresponding heteromultimer involved in interallelic complementation, the damaged regions are not symmetrically related. In the tirst case the t,wo identical symmetrically related damaged sites reinforce one another, in the second case (in the hybrid protein) the two different damaged sites do not necessarily work in the same direction. For this reason alone a, hybrid protein is more likely to be active. In other words on this basis alone one would expect at least some interallelic complement&ion. (Arguments of this kind are used by Monod et al., (1965) in discussing the evolutionary advantages of multimeric proteins.) Finally, it seems possible that the ideas outlined here, depending on the distribution of specific binding sites, allosteric transitions end overall quaternary &-ucture, might be open to experimental test by other than detailed X-ray studies. Note added in proof. A paper by Perutz & Lehmann on the Molecular Pathology of Human Haomoglobin (Perutz, M. F. & Lehmann, H. (1968). Nature, 219, 902) which has just appeared, seems to be of interest in relation to the ideas put forward above. The mlltants discussed by Perutz & Lehmann tend to fall into groups associated with active sites (haem groups) or with parts of the structure associated with conformational change (contacts between subunits), in other words with groups of t,he kind considered above.
242
8. MoGAVIN
The fact that the twofold axis in haemoglobin runs through a hole in the structure makes homologous correction in the vicinity of the twofold axis unlikely in this csse. It is possible to argue that there may be e tendency for all proteins to evolve in such a way that axes of rotational symmetry run through holes. If they do then homologous correction is likely to be rare. These points seem of interest irrespective of whether haemoglobin shows effects of the complementation type or not. It is intended to develop these points in another paper. Thanks sre due to Dr D. Stansfield of the Department of Biochemistry, Dr H. R. Wilson of the Department of Physics and particulerly to Dr A. C. Hastie of the Department of Botany of this University, for discussion. Thanks are also due to Dr Hastie for introducing me to the subject. Isotopes Laboratory University of Dundee Dundee, Scotland Received 23 May 1908, and in revised form 3 July 1968
STEWABT MCGAVIN
REFERENCES Criok, F. H. C. & Orgel, L. E. (1964). J. Mol. Bid. 8, 161. Finoham, J. R. 5. (1966). &net& C~lementcrtion, New York: Benjamin. Gillie, 0. J. (1966). &net. Rec. Cad. 8, 9. Kapuler, A. M. & Bernstein, H. (1963). J. Mol. Biol. 6, 443. Monod, J., Wyman, J. & Changeux, J. P. (1966). J. MOE. Biol. 12, 88.
Interallelic Complementation and Allostery The defects responsible for loss of activity in mutant forms of enzymes need not always involve the active sites themselves and could involve parts of the peptide oh&n associated either with, or with the conditioning of, conformational change. The point of this letter is to show that if this is so, then extensive complement&ion might result without the need for homologous correction. It is fairly generally accepted that interallelic complementetion is something which results from the interaotion of subunits in multimeria proteins. (For recent reviews e.eeFinaham, 1966, and Gillie, 1966.) The most plausible theories of the effect-those of Kapuler & Bernstein (1963) and of Crick & Orgel (1964)-hsve involved homologous oorrection. It has been assumed in other words that the close juxtaposition of a defeotive region in one monomer with a correct homologous region of another monomer can result in the correction of the defect. As pointed out by Crick & Orgel the models of protein structure invoked by Kepuler b Bernstein are implausible. Crick & Orgel themselves suggest that multimerio proteins are likely to be symmetrical and in particular that they are likely to involve rotation axes. They also suggest that correction is most likely to take place in the vicinity of the rotation axes. As Crick & Orgel point out, their theory does lead to the general type of relationship observed in oomplementation. In what follows it is eccepted that multimeric proteins are likely to be symmetrical in the way that C&k & Orgel suggest. Crick t Orgel dismiss 8s a special case complementation between monomers where the active site is made up of parts of two or more subunits. (This special c&se is shown diagrammatically in Figure 1). They consider that this may be oorrect in some cases but that it can hardly provide a basis for a general theory of the approximate linearity of complement&ion maps spreading over considerable lengths. Thus with the arrangement of Figure 1 all the complementing mutants would fall into two groups, (see also Fincham, 1966). It seems, however, that the mechanism of this speoial ease, simple juxtaposition of parts of active sites, unlike that of homologous oorreotion, is one which simply must work. It therefore seemed worth asking if there are any ways in which it might be extended. Another reason for looking for some general explanation of complement&ion in addition to the homologous correction one of Crick & Orgel, is that complement&ion maps are often basically simple. Also, (see for example Gillie, 1966) in many cases a high proportion of mutants show complement&ion, whioh is diffioult to account for on the basis of homologous correction in the vicinity of a.xes of symmetry. It seems possible to extend the simple juxtaposition theory (that of Fig. 1) by considering that an enzyme might be rendered inaative by damage to sny one of several sites, possibly its allosterio binding sites. The simplest ease of this kind involves a dimerio allosteric enzyme, eeoh monomer of which has two sites, 8 and A (possibly substrtlte and activator binding sites) each complete in itself. If it is assumed that for activity all that the dimer requires is one aotive A site and one adive S site, then this 239
S. McGAVIN
240
FIG. 1. Diagram of the simple “special case”. If both A’s or both B’s are damaged the dimer is inactive. If an A in one monomer and a B in the other are damaged the dimer is active since it has one undamaged AB site.
(a)
’ A,(b)
I I ,f
I -- A
s
S'
-I-
'
S'd
SA AS'
-+----
A'
S
I II ---v--I I
I I
FIG. 2. (a) Diagram of an enzyme structure consisting of two identical peptide chains related by a twofold axis of rotation. The structure has two sites (SS’ and AA’) essential to its activity. In active site SS’, S is considered to come from one subunit and S’ from the other and in AA’, A is considered to come from one subunit and A’ from the other. SS’ and AA’ might be substrate binding and activitor binding sites. The structure is shown as a cylindrical development. The relationship of S and A at the left of the diagram to S’ and A’ at the right is the same as the relationship of S and A to S’ and A’ at the centre. (b) Complementation map resulting from the model of Fig. 2(a). The map can be folded into a circle. TABLE
1
Compkmentation properties of the model shown in Pig. 2(a)
S S’ A A’ SS’ AA’ SA SA S’A S’A’
S
S’
A
A
SS’
AA’
0 + + + 0 0 0 0 + +
+ 0 + + 0 0 + + 0 0
+
+ + + 0 0 0 + 0 + 0
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
SA
SA’
S’A + 0 0 + 0 0 0 + 0 0
S’A’ + 0 + 0 0 0 i0 0 0
S, S’, A and A’ are groups of mutants which make half sites S, S’, A and A’ inactive. SS’, AA’, etc. are groups of mutants which make pairs of half sites S and S’, A and A’, SA etc. within one monomer inactive. + indicates that groups of mutants could complement. 0 indicates that groups of mutants would not complement.
LETTERS
TO
THE
EDITOR
241
results in complement&ion properties similar to those of the “special case” discussed above (Fig. 1). If, however, each site is made up of parts of the two subunits, then the number of complementing groups can increase considerably. This is illustrated by means of a model shown in Figure 2(a) and in Table 1. There are now two kinds of groups of complementing mutants, one kind affecting only one-half site, the other affecting two different half-sites within a monomer. Mutations affecting three half sites would not complement in this case. The resulting complement&ion map involving four units and eight groups is shown in Figure 2(b). The number of complementing groups can be further increased and the map made more complex if damage to a third site, again consisting of two half sites, could render the enzyme inactive. In this case some mutations affecting three half sites will complement. One such system, for example, can result in a map involving six units and twenty-six complementing groups, eighteen of which, those most likely to occur, can be included in a linear map. The pattern of complementation could be simplified in either of these cases if some of the specific sites were complete within monomers. It could be made more complex if sites are contributed to by more than two monomers, or if more than three sites are essential to activity. So far defects associated with specific binding sites have been considered as examples. Allosteric enzymes, however, are presumably structures which are delicately balanced between two or more states (see for example Monod, Wyman C%Changeux, 1965), damage to many different sites within or on an allosteric protein could perhaps interfere with its moving from one state to another or could lock it in one state. It seems possible therefore that mutations not associated directly with any of the specific binding sites might be able to tip the balance one way or the other and that this loss of balance might be corrected by another mutant thus giving rise to further complement,ing groups. If mutations could cause the loss of, or the correction of a rather delicate balance then it seems that “bad correcting bad” might be involved. An important point here which bears particularly on mutations affecting conformational changes, rather than the active sites themselves, is that in a symmetrical mutant homomultimer the damage is also symmetrical, in the corresponding heteromultimer involved in interallelic complementation, the damaged regions are not symmetrically related. In the tirst case the t,wo identical symmetrically related damaged sites reinforce one another, in the second case (in the hybrid protein) the two different damaged sites do not necessarily work in the same direction. For this reason alone a, hybrid protein is more likely to be active. In other words on this basis alone one would expect at least some interallelic complement&ion. (Arguments of this kind are used by Monod et al., (1965) in discussing the evolutionary advantages of multimeric proteins.) Finally, it seems possible that the ideas outlined here, depending on the distribution of specific binding sites, allosteric transitions end overall quaternary &-ucture, might be open to experimental test by other than detailed X-ray studies. Note added in proof. A paper by Perutz & Lehmann on the Molecular Pathology of Human Haomoglobin (Perutz, M. F. & Lehmann, H. (1968). Nature, 219, 902) which has just appeared, seems to be of interest in relation to the ideas put forward above. The mlltants discussed by Perutz & Lehmann tend to fall into groups associated with active sites (haem groups) or with parts of the structure associated with conformational change (contacts between subunits), in other words with groups of t,he kind considered above.
242
8. MoGAVIN
The fact that the twofold axis in haemoglobin runs through a hole in the structure makes homologous correction in the vicinity of the twofold axis unlikely in this csse. It is possible to argue that there may be e tendency for all proteins to evolve in such a way that axes of rotational symmetry run through holes. If they do then homologous correction is likely to be rare. These points seem of interest irrespective of whether haemoglobin shows effects of the complementation type or not. It is intended to develop these points in another paper. Thanks sre due to Dr D. Stansfield of the Department of Biochemistry, Dr H. R. Wilson of the Department of Physics and particulerly to Dr A. C. Hastie of the Department of Botany of this University, for discussion. Thanks are also due to Dr Hastie for introducing me to the subject. Isotopes Laboratory University of Dundee Dundee, Scotland Received 23 May 1908, and in revised form 3 July 1968
STEWABT MCGAVIN
REFERENCES Criok, F. H. C. & Orgel, L. E. (1964). J. Mol. Bid. 8, 161. Finoham, J. R. 5. (1966). &net& C~lementcrtion, New York: Benjamin. Gillie, 0. J. (1966). &net. Rec. Cad. 8, 9. Kapuler, A. M. & Bernstein, H. (1963). J. Mol. Biol. 6, 443. Monod, J., Wyman, J. & Changeux, J. P. (1966). J. MOE. Biol. 12, 88.