Regulatory control through conformation changes in proteins

REGULATORY CONFORMATION

CONTROL

THROUGH

CHANGES

IN

PROTEINS

D. E. KOSHLAND, JR. Department of Biochemistry, University of California, Berkeley, California

THE regulation of metabolism can occur in a number of ways. The synthesis of enzymes may be affected so enzyme production is increased or stopped. A direct competition at the active site may occur between two different metabolites or an indirect interaction may occur which changes the conformation of a protein in order to regulate its function. In previous papers from our laboratory various experimental and theoretical aspects of these conformational phenomena have been explored. It seemed appropriate, therefore, at a symposium of this sort to emphasize general relationships which, appear to be emerging from these studies. For detailed mathematical and experimental developments the original papers should be consulted. In Fig. 1 is shown the schematic illustration of the interaction of ligands with a flexible enzyme. If the ligand has all the proper features it will induce a conformation change to bring the ~atalytic groups A and B into proper alignment to cause reaction. Another molecule having the proper bond to be broken but incapable of inducing the proper alignment of catalytic groups will fail to react. This "induced fit" theory was initially designed to explain the anomalies in the literature such as the failure of molecules smaller than the substrate to react when it was clear that they had access to the enzyme surface(I,2). One of the most important consequences of this theory was the role it allowed for small molecules which were not consumed in the reaction to influence the velocity of the reaction(3). In Fig. 2 is shown a schematic illustration of such interactions showing the role of activators which may help induce the proper alignment even though they are not bound at the site of the substrate molecule. Thus a "deficient substrate", i.e., one that lacks the necessary bulk for buttressing the protein conformation appropriately, may be made adequate by the binding of a second molecule as shown in the figure. Examples in isolated systems for just such phenomena have been demonstrated, e.g., the work on trypsin by Inagami and co-workers(4). In the 291

292

D. E. KOSHLAND, JR.

N-SUBSTRATE~

. . . . . . .. ~ '

FIG. 1 Schematic illustration of induced fit theory. Upper lea: Protein in absence of substrate. Upper right: Protein after substrate has induced conformation change bringing catalytic groups A and B into proper alignment. Lower: Pretein with non-substrates which are too small or too large to bring catalytic groups into proper alignment.

" f e e d b a c k " systems it has been found that an entirely new regulatory site designed specifically for the end product of a metabolic chain has a particularly valuable biological function since it can "turn off" the first e n z y m e in the chain(5,6). Most o f the e n z y m e s which are involved in regulatory behavior are composed of subunits and exhibit cooperative effects. A normal MichaelisMenten binding gives a hyperbolic curve on a ~ (fraction saturation) vs. ligand concentration plot and all such curves require an 81-fold change in ligand concentration to go from 10 to 90% saturation of the binding sites of the protein. In a number of proteins a sigmoid curve is obtained in such a y vs. (x) plot and ony a 3- or 6-fold increase in ligand concentration is needed to go from 10 to 90% saturation. This p h e n o m e n o n has been called the "cooperative effect" b e t a u s e it was recognized theft the first

REGULATORY CONTROL THROUGH CONFORMATION CHANGES

NON-COMPETITIVE-IF (B) INVOLVED IN CATALYSIS AND NOT BW~DING COMPETITIVE-IF (B) ESSENTIAL TO BINDING

293

ACTIVATOR X STABLIZING ACTIVE CONFORMATION FOR COMPOUND WHICH WOULD NOT INDUCE PROPER CONFORMATION IN ABSENCE OF X.

Fro. 2 Protein in which small molecules not metabolized themselves can affect conformation change. Left-inhibitor, I, prevents proper conformation. Right-activator, X, helps stabilize proper conformation.

molecule of ligand must somehow help the second molecule of ligand to bind. To explain these cooperative effects we have proposed a theory which follows closely on the type of arguments used in the induced-fit theory (7). In essence it is assumed that the bound ligand induces a conformation change in one subunit which may change the stabilization energy and/or shape of the neighboring subunits. Because of this change in shape it is possible for subsequent molecules of ligand to bind more readily than the first. These models and their ramifications have been discussed extensively elsewhere(7-10). It seemed appropriate in this symposium to examine some of the general principles behind the findings on a specific protein and to illustrate how our knowledge of protein structure enables us to understand this facet of regulatory control. In Fig. 3 the changes in the conformation of isolated and polymerized subunits are illustrated against an energy scale. We shall designate the shape of the subunit in the absence of any ligand with a circle or the letter A. In Part a of Fig. 3, the binding of ligand S is shown to induce a conformation change here designated by a square or the letter B. Obviously, the protein could undergo a spontaneous conformation change to conformation B, but the energy required is so great that there will be very few molecular species present in the B conformation in the absence of bound ligand. If the substrate binds strongly and preferentially to the B conformation, it will "pull" the equilibrium and cause conversion of A species to

294

D. E. K O S H L A N D , JR.

[]

®

Free Energy

0

®

rE~ (A)

(B)

CO)

FIo. 3 Energy changes affected by subunit interactions. Part (a). Conformations in absence of figand (circle and square) have energies such that little of the "square species" (also called conformation B) will exist in the presence of the circles (designated as conformation A). Ligand binds preferentially to conformation B stabilizing it with respect to conformation A: Part (b). Same as part (a) except that subunit interactions stabilize adjacent squares. Part (c). Activator and inhibitor effects illustrated (see text).

BS species. In other words, the very strong affinity of the ligand for the new conformation overcomes the unfavorable energy barrier of producing the change in shape. It is not necessary, of course, that the change in shape occurs in exactly this manner. Ligand could be bound first to the unchanged conformation and then cause a series of isomerizations leading to the final square conformation equally satisfactorily(l 1). However, we have designated the changes in this manner because they might occur in this way and because it is easier to illustrate the energetic considerations involved. If we now imagine this same subunit or monomeric unit adjacent to a second subunit the energetics of its reaction may well change. If adjacent squares have net attraction energy greater than that of an adjacent square and circle, the change in energy in the circle-to-square transition in the dimer will be less than in the isolated monomer. Conceptually we could also break this process up into two steps, i.e., an initial change of the circle to the square equal in energy to that in Fig. 3a followed by a second step in which the separated square associates with the square already containing bound S with a consequent decrease in free energy. If the binding of substrate to the square conformation is equal in any state of aggregation, we see the overall free energy change for the change from A to BS is greater in the dimer. In Fig. 3c we can now use this example to explain the action of activators or inhibitors. The ligand X here could be any compound which

REGULATORY CONTROL THROUGH CONFORMATION CHANGES

295

induces a conformation change similar to that shown by the substrate in Figs. 3a and 3b. If the substrate is at a low concentration relative to the Michaelis constant (the usual situation in the cellular milieu) then a ligand of this type would serve as activator since it would increase the concentration of ES complex and hence increase the metabolism of the substrate. Ligand L is shown inducing a different type of conformation change (shown here by a hexagon) which distorts the neighboring subunit to a similar hexagon. If this conformation cannot bind substrate, then the inhibitor L acts by stabilizing a conformation which cannot function. To this point we have discussed stabilizing conformations without discussing how they might occur. In Fig. 4 the manner in which such energy

Na+CI CI-

Na +

CI-

No +

Na +

CI-

Na+CI No + CICl+

Na +

No + _

CI-

Cr

.. ÷ NO

Na +

FIG. 4 Schematic illustration of manner in which induced conformation changes can stabilize protein structures. Top line shows subunits held together by hydropbobic bonds. Induced conformation change brings+ a n d - groups closer together in circle-square intermediate and very close in adjacent squares. Counter-ions in medium, e.g., Na + and CI-, can nullify effects of charges unless they are very close. Second line shows same effect of activator X and inhibitor L in protein containing non-identical subunits or a single peptide chain.

changes might be effected is shown schematically. A hydrophobic bond (H) and electrostatic attractions (+ and --) are used to illustrate these phenomena but hydrogen bonding, electro}tatic repulsions and steric hindrance can, of course, also occur in actual cases. In the top part of the figure, an example of two identical subunits (both circles) is considered.

296

D. E. KOSHLAND, JR.

The subunits are held together by a hydrophobic bond, the electrostatic charges being too far apart to have any appreciable force. On binding X the conformation change brings the ÷ and - c h a r g e s closer together. If they are still too far apart to have an appreciable interaction, the adjacent square-circle will have essentially the same interaction energy as the circle-circle, i.e., in our mathematical nomenclature KAB ~- K4A" If they are close enough then there will be added stability and KAn > KAA. In either case the binding of the second molecule of X brings the two charges into close juxtaposition so K~B > KAB >! KAA- Thus the conformation change which places adjacent squares together has a net added free energy of stabilization as illustrated in Fig. 3. In the second line, this same process is illustrated for a single polypeptide chain. In this example the hydrophobic bond holding the subunits together in the top line is replaced by the covalent bond of the peptide chain but the non-covalent interactions between chains are identical. This is just to illustrate that regulatory effects can operate equally well through interactions between sites on the same subunit as between adjacent subunits. In the lower part of the figure similar relationships are demonstrated for non-identical subunits with two different ligands. The new conformations induced by X and L need not be the same and yet both may be stabilized relative to the initial circle and triangle conformations existing in the absence of bound ligand. If the conformation induced in the initially triangular subunit is active in the form induced by X it will be an activator. If it is inactive in the form induced by L it will be an inhibitor. In the lower line the same phenomenon is demonstrated for a single polypeptide chain. In both of these examples Na ÷ and C1- are inserted to emphasize that the solution contains counter-ions which neutralize most charges on a protein unless the charges are in close juxtaposition. These ions were omitted from the upper two lines for simplicity in presentation only. In Fig. 5 a number of changes of conformation in a dimer are illustrated. If circles designate the conformations of the subunits in the unliganded state and squares the conformation in the fully liganded state, intermediate states can show various types of conformation changes. At the top we show the very extreme case of one subunit in the square and one in the circle. As discussed above the change in conformation of one subunit can lead to cooperative effects by changing the stability of the neighboring subunit even though the shape of the neighbor is not changed immediately. An extensive treatment of such a "two conformation" model has already been published (7-9). The change in conformation on binding S may change the shape as well as the stability of the neighboring subunit. It may distort it partially to a

REGULATORY CONTROL THROUGH CONFORMATION CHANGES E

ES

297

ES2

FIG. 5 Possible effects of induced conformation change on dimer composed of identical subunits. Circles represent unliganded conformations, squares conformations when protein fully saturated. Intermediates include examples in which one subunit completely changed (top three lines) with neighboring subunit unchanged (top line), partially changed (second line) or completely changed (third line). Bottom three lines show cases in which liganded subunit conformation is intermediate between circle and square and neighboring subunit is unchanged or partially changed.

conformation which is different from the initial or final states or completely to the "final" square conformation as shown in the second and third lines of the figure. Moreover it is not necessary that the liganded subunit be completely changed to the square conformation on binding S. The interactions of the second subunit may prevent the immediate change to a square conformation on binding of the first S. The resultant "drag" of the second subunit may thus cause intermediate situations of the type shown in the lower three intermediates in that figure. Evidence that such intermediate structures exist has already been obtained in the case of rabbit muscle glyceraldehyde-phosphate-dehydrogenase(10). As one looks at the many alternatives of Fig. 5 it is inevitable to desire some simplifying assumptions. Two simplifications have already been proposed. One by Monod, Wyman and Changeux(12) suggests that there are symmetry requirements for all proteins containing subunits. This requirement leads to the corollary that a change in the conformation of one subunit leads to an equivalent change in all the identical subunits. A

298

D. E. K O S H L A N D , JR.

further symplifying assumption is made that there are only two or a few states of the protein. The only intermediate forms which would satisfy the symmetry requirement would be the third intermediate from the top and the intermediate in the last line of the Figure. The last intermediate would be eliminated if one considered only two final states for the protein. Koshland, Nemethy and Filmer have considered a sequential model which is in essence very general but the model was simplified with two restrictive assumptions (a) there are only two subunit conformations and (b) that only subunits with bound ligand undergo a conformation change. The only intermediate forms in accordance with the first assumption are at the top and the third from the top lines of the Figure and the only form in accordance with both assumptions is on the third line. In previous papers from Monod's laboratory and Ours and many other individuals these simple models have been used to explain at least qualitatively a number of regulatory enzyme systems. Moreover, general considerations of these simplest models indicate that they may be excellent first approximations to the data in some cases which will ultimately require more complex interpretations. Until further studies are performed it is impossible to say whether the simplest models will be adequate in most cases or whether the interactions will be very complex. Studies on yeast glyceraldehyde dehydrogenase by Eigen et al. seem to follow the simplest Monod model(13) and studies on hemoglobin by Ogawa and McConnell(14) seem to agree with our simplest sequential model. However, the studies on rabbit muscle glyceraldehyde-3-phosphate dehydrogenase establish that in the case of at least one enzyme neither of these two very simplest models will apply (10). From these studies and general considerations of protein structure it seems probable that the permutations of Fig. 5 will all eventually be found and that the more general approach to subunit interactions will be required. Studies along these lines have led us to the following tentative conclusions: a. The treatment of more general models involving various species as shown in Fig. 5 is more laborious but not necessarily much more difficult than the simplified models involving just a few conformations. The simplified models involve only interactions of two subunit types AA, AB and BB whereas it will be necessary to consider additional conformations C, D, E, and additional subunit interactions, BC, AB, CD, DE etc. in the more general models. However, the mathematical methods developed for the simplest sequential m o d e l s ( 7 - 9 ) a r e readily extended to more complex models and the basic energetic features discussed above are equally applicable to these more complex situations. The tools for detecting minor changes in conformation will need to be sharpened to dis-

REGULATORY CONTROL THROUGH CONFORMATION CHANGES

299

tinguish between specific details but conceptually the problems are not more difficult. b. The general considerations of a sequential model involving multiple conformations allow us to place the behavior of regulatory proteins into a general framework which is qualitatively very satisfying. Subunit interactions are basically the same as interactions within a polypeptide chain. The binding of a ligand induces a new conformation which changes the stability relationships and/or the shape of neighboring subunits or of neighboring parts of the same polypeptide chain. This binding therefore will inevitably affect observed phenomena such as the binding of a second ligand or the activity of the protein. c. The binding of one ligand which induces a conformational change may make the affinity of a second ligand greater or less. If the two ligands are different, then the ligand which makes substrate bind better is called an activator and the ligand which makes substrate bind less is called an inhibitor. If the ligands are the same, and the first molecule of substrate bound increases the affinity of the second, we say that a cooperative effect is observed. If the first substrate decreases the binding of the second molecule of substrate we call it a negative-cooperative effect. Thus, we see that the mutual effect of like ligands is no different conceptually than the mutual effect of unlike ligands. Interactions which affect activity as well as binding affinity would also be expected to have parallel consequences. Thus cooperative and anti-cooperative effects, allosteric inhibitors and activators all become part of a simple general picture mediated by conformational changes. d. The variety of interactions observed in enzymes can then be readily explained by the variety in the nature of intra- and inter-chain interactions. If there is very little or no change in the interactions of subunits on binding of the first ligand, Michaelis-Menten behavior will be observed even though the subunits are held together in a polymeric protein. Each subunit would be acting independently even though it is present in a polymer because of the weak coupling between subunits. The extent of coupling of subunit interactions could then vary over a wide range so that each of the permutations of Fig. 5 would be conceivable under different circumstances. Evolution could then have operated as a giant computer varying the strengths of subunit interactions and the energy of the conformation changes until the kinetic behavior necessary for regulatory control is selected. e. It seems probable that no single assumption such as "symmetry" requirement or a "one ligand-one subunit change" is likely to be generally true although they may be excellent first approximations

300

D. E. KOSHLAND, JR.

in many cases. Rather, it seems likely that there is a mutual interaction between protein and ligand to induce a conformation which is kinetically and thermodynamically optimum for that complex. Several ligands may induce the same conformation or the same alignment of catalytic or binding groups may be effected in different ways. In general however, each ligand-protein interaction has the potential for a new structure and many such changes will be observed. The polymerization of subunits leads to inter subunit interactions which may amplify or dampen the effects of the metabolite concentration changes. A 2-5-fold change in concentration in a cooperative system may have the effect of an 80-100-fold change in a monomeric system. An 80-100-fold change in an anti-cooperative system may have the effect of a 5-10-fold change in a Michaelis-Menten system. In some systems increased sensitivity may be desirable; in others, a resistance to regulatory control may be essential. Subunit interactions provide possibilities for either alternative. g. Finally, the phenomenon which ultimately affects the enzyme action is the conformation change in the protein. It is possible to imagine such a conformation change being induced by compounds other than the normal metabolites or by agents other than small molecules. Carcinogens and drugs come to mind in the first category. The interactions of cell surfaces, the effects of pressure and hormones seem pertinent in the second category.

SUMMARY

Cooperative effects in proteins such as hemoglobin and feedback enzymes have been observed extensively. A sequential model to explain these effects has been developed in which it is assumed that a ligand can induce a conformational change in an individual subunit and that the distortion in this one subunit may be transmitted with varying efficiencies to neighboring subunits. Expressing these interactions in mathematical form it is found that the models can explain qualitatively and quantitatively a wide variety of phenomena observed in the biological literature. Key features of the model are (a) that there are sequential changes in conformation as ligand is bound, i.e., hybrid conformational states can exist, (b) that the efficiency of transmission of the effect in one subunit to neighboring subunits may vary widely, i.e., the coupling may be very low in which case the protein will exhibit Michaelis-Menten kinetics or it may be very high in which case highly cooperative phenomena will be observed, and (c) that the final conformational state depends on the pro-

REGULATORY CONTROL THROUGH CONFORMATION CHANGES 301 tein and the ligand bound, i.e., there may be a considerable diversity in the final conformational states with different ligands.

ACKNOWLEDGMENT

The author expresses his gratitude to the Public Health Service (NIH Grant AM-09765) and the National Science Foundation (Grant GB-4186) for their generous support of this research.

REFERENCES 1. D. E. KOSHLAND, JR., Application of a theory of enzyme specificity to protein synthesis, Proc. Natl. Acad. Sci., U.S. 44, 98-104 (1958). 2. J. A. YANKEELOV,JR. and O. E. KOSHLAND,JR., Evidence for conformation changes induced by substrates of phosphoglucomutase, J. Biol. Chem. 2411, 1593-1602 (1965). 3. D. E. KOSHLAND,JR., Enzyme flexibility and enzyme action, J. Cell. Comp. Physiol. 54, Supplement 1,245-258 (1959). 4. T. INAGAMI and T. MtmACHI, The mechanism of the specificity of trypsin catalysis. III. Activation of the catalytic site of trypsin by alkylammonium ions in the hydrolysis of acetylglycine ethyl ester, J. Biol. Chem. 239, 1395-1401 (1964). 5. J. C. GERHART and A. B. PARDEE, The enzymology of contol by feedback inhibition. J. Biol. Chem. 237,891-896 (1962). 6. J. MONOD, J. P. CHANGEUX and F. JACOB, Allosteric proteins and cellular control systems, J. Mol. Biol. 6,306-329 (1963). 7. D. E. KOSHLAND, JR., G. NEMETHY and D. FILMER, Comparison of experimental binding data and theoretical models in proteins containing subunits, Biochemistry 5, 365-385 (1966). 8. i . E. KIRTLEY and D. E. KOSHLAND,JR., Models for cooperative effects in proteins containing subunits. Effects of two interacting ligands, J. Biol. Chem. 242, 4192-4205 (1967). 9. J. E. HABER and D. E. KOSHLAND,JR., Relation of protein subunit interactions to the molecular species observed during cooperative binding of ligands, Proc. Natl. Acad. Sci., U.S. (in press). 10. D. E. KOSHLAND,JR., A. CONWAY and M. E. KIRTLEY, Conformational changes and the mechanism of action of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase, Symposium o f Federation o f European Biochemists, Academic Press (in press). 11. D. E. KOSHLANO,JR. and M. E. KIRTLEY, pp. 217-249 in Major Problems in Developmental Biology (M. LocKI, ed.) Academic Press, New York (1967). 12. J. MONOD, J. WYMAN and J. P. CHANGEUX,On the nature of allosterie transitions: A plausible model, J. Mol. Biol. 12, 88-118 (1965). 13. K. KIRSCHNER,i . EIGEN, R. BITTMAN and B. VOIGHT, The binding of nicotinamideadenine dinucleotide to yeast o-glyceraldehyde-3-phosphate dehydrogenase; Temperature-jump relaxation studies on the mechanism of an aliosteric enzyme, Proc. Natl. Acad. Sci., U.S. 56, 1661-1667 (1966). 14. S. OGAWA and H. M. MCCONNELL,Spin-label study of hemoglobin conformations in solution, Proc. Natl. Acad. Sci., U.S. 58, 19-26 (1967).