1 Laboratory of Experimental and Computational Biology and 2 Intramural Research Support ProgramSAIC, Laboratory of Experimental and Computational Biology, NCI-FCRDC, Bldg 469, Rm 151, Frederick, MD 21702, USA and 3 Sackler Institute of Molecular Medicine, Department of Human Genetics and Molecular Medicine, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
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Abstract |
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Keywords: amino-terminus/chaperones/energy landscape/folding funnels/proregions/prosequences/protein binding/protein folding
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Introduction |
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Binding and folding are similar processes, governed by similar principles (Tsai et al., 1998, 1999a
). Consistently, a chaperone can be either a separate molecule or chain-linked. Both have been documented extensively. Different classes of molecular chaperones exert their effect in different ways, either by temporarily hosting the misfolded proteins in their cavities or by binding to hydrophobic patches on their surfaces (Ellis, 1997
, 1998
; Netzer and Hartl, 1998
; Ellis and Hartl, 1999
). Previously, it has been suggested that molecular chaperones act through direct catalysis of the folding proceses. However, recently it has been shown that the action of the chaperonins might be by preventing misfolding or by forcing unfolding of a protein trapped in a misfolded conformation (Walter et al., 1996
; Shtilerman et al., 1999
). These chaperones have no effect on the final structure of a protein. In contrast, the chain-linked molecular chaperones have been proposed to act by conveying steric information that is essential for correct folding (Shinde et al., 1997
). However, subsequently, the fragment playing this role is cleaved off. Hence, in this sense, in all three cases the chaperone molecules or the relevant fragments act in a similar way, namely, via transient binding. Nevertheless, there is a difference between the molecular chaperones and the fragment chaperones: in the first case, the molecular chaperones are believed to aid through binding to intermediate states, to prevent misfolding, with subsequent release. On the other hand, in the intramolecular chaperone proregion, the pro-fragment is attached to the polypeptide chain until the chain achieves its final, native conformation. Only then it is self-cleaved.
Here we propose a class of intramolecular chaperone-like fragments, those that are chain-linked and uncleaved. These, permanently linked intramolecular fragment chaperones recur frequently in proteins. While they have not been recognized as such, perhaps owing to the fact that they remain attached to the molecule throughout its lifetime, Shinde and Inouye (1994) have searched for sequence similarity between motifs they have identified in prosequences and other, related and unrelated proteins, looking for potential significance. Here we argue that such a permanently linked intramolecular chaperone is a building block of the protein structure (Tsai et al., 1999a,b
; Kumar et al., 2000
), is frequently (although not necessarily, as discussed below) located at the amino-terminus of the molecule and is essential for obtaining the native conformation. If it is chopped off, the remainder of the molecule may still attain a stable structure, but it will be non-native. This type of (usually) amino-terminal fragment is in contact with other building blocks of the structure, mediating their interactions. In its absence, these building blocks are still likely to be in their native conformations, but their association is non-native. We further argue that this type of intramolecular chaperone-like building block is likely to be more frequently observed in cases of non-sequential folding. In such cases, it serves to mediate the interactions between the other building blocks, preventing their potential non-native contacts. Such an amino-terminus building block fragment is then likely to be in contact with a number of building blocks. For example, it may associate with the carboxy-terminus building block and/or concomitantly with other, internal blocks. For clarification, Figure 1
presents a schematic drawing illustrating an uncleaved, `hidden', intramolecular chaperone-like fragment. The figure further illustrates the consequences of either its absence or of its being flipped out and misfolded. Either way, under such circumstances, stable, but non-native interactions between the remaining building blocks take place. In contrast, if other building blocks are absent, the population time of the remainder of the native structure is still the highest among all (non-native) alternative conformations.
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Below, we first describe the intermolecular chaperones and the intramolecular, proregion chaperone fragments which undergo proteolysis after the molecule attains its native state. Next we describe our proposed intramolecular, uncleaved chaperone-like building blocks. We review current literature, showing it to be consistent with our uncleaved intramolecular chaperone-like building block proposition.
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Intermolecular and intramolecular chaperones: the need for guidance and catalysis in protein folding |
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Most of the chaperones known to date are distinct molecules whose role is to prevent misfolding. Among these, two families have been studied particularly well. The first is the chaperonins, which include the GroEL, and the second is the smaller chaperone proteins (Ellis, 1998; Netzer and Hartl, 1998
; Ellis and Hartl, 1999
). The mechanism differs between these two families. The chaperonins are large, multi-subunit allosteric proteins, with central cavities into which the misfolded proteins enter, with subsequent ejection (Shtilerman et al., 1999
). On the other hand, molecular chaperones belonging to the second family appear to act via binding to the surface of misfolded proteins, at sites with an extensive exposed non-polar area. This second family consists of small proteins and hence cannot have a cavity large enough to hold the misfolded proteins. Both the chaperonins and most of the small molecule chaperones apparently do not recognize specific sequences or well-defined structures. This mostly sequence non-specificity in binding is in contrast to the chain-linked, sequence- and structure-specific intramolecular chaperones discussed below. Among the few exceptions, cases such as that of the bacterial lipase limA chaperone (Hobson et al., 1993
) which is separated from the lipase (lipA) by only three nucleotides and is lipase-specific, can be considered as evolutionarily related to the intramolecular proregion chaperones.
The third family is the cleaved-off intramolecular chaperones. This is the class that Ellis has aptly christianed `steric chaperones' in his recent insightful mini-review (Ellis, 1998). It is well known that some proteins are synthesized with an extra fragment at their amino termini. This fragment is essential for correct folding (Baker et al., 1992
). However, after the protein folds, these fragments are degraded. Such cases typically occur in serine proteases, such as subtilisin (Figure 2a
),
-lytic protease (Figure 2b
) and aqualysin from bacteria and carboxypeptidase Y from yeast. These fragments act as inhibitors, covering the active sites of the enzymes, hence it is essential that they be cleaved and digested for the enzyme to be functional. However, if the fragment is cleaved off in the construct, prior to protein synthesis, the newly synthesized chain misfolds. On the other hand, if the cleaved off fragment is mixed with the remainder of the chain in solution, a correctly folded protein is obtained. If the fragment is added to a solution containing an already misfolded chain, the chain converts to its native fold. Hence, as pointed out by Shinde and Inouye (1994), there is a major difference between the intermolecular and intramolecular proregion chaperones. In the former case, in the absence of the chaperone, under proper conditions, the protein molecule would still fold into its native state, albeit with reduced efficiency. This, however, is not the case for intramolecular proregion fragments. In their absence, the protein will misfold.
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The kinetic and thermodynamic roles of proregions in protein folding |
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The function of a prosegment is to ensure that the protease functions at the right place and time. Often, a pro-region has no role in folding; rather, the mature sequence already contains most of the information required to specify its native three-dimensional conformation (Price-Carter et al., 1996; Veldhuizen et al., 1999
). The folding-related roles of the proregion may be classified into two types of cases: In the first, the proregion acts as an intramolecular chaperone to overcome kinetic barriers in folding. In the second, the proregion acts as a thermodynamic stabilizer, through uncleaved disulfide bonds.
Over the last few years, Agard and co-workers have made a number of profound observations on proregion chaperones, particularly on -lytic protease. They have shown that the stability of the enzyme derives not from thermodynamics, but from its kinetics. The proregion is the means through which this is achieved. The co-evolution of this region facilitates folding into the native state, which is not the most stable one. However, the barrier separating it from the global minimum is high enough to keep the
-lytic protease in its native, functionally active state (Baker et al., 1992
, 1993
; Cunningham et al., 1999
). Specifically, the studies of Sohl et al. (1998) have indicated that the proregion catalyzes protease folding by directly stabilizing the folding transition state. Anderson et al. (1999) have investigated the precursor, the complexed structure of the proregion with the mature
-lytic protease enzyme and the isolated proregion. They have shown that there are substantial similarities in the secondary structures of the precursor and the complex, but they differ in their tertiary structures and in particular in their stability. Correlation with the proregion has indicated that it is fully folded and stabilizes the structure of the enzyme. The rate of folding of the precursor was shown to be biphasic, with the fast phase dependent on the rate of the proregion folding. Hence the proregion folds first, with subsequent catalysis of the folding of the protease domain (Anderson et al., 1999
). On the basis of these observations, Agard and co-workers have proposed a model in which the major role of the proregion is to bind to the ß-hairpin at the C-terminal domain of the
-lytic protease, to form a five-stranded ß-sheet and thereby to position the ß-hairpin correctly, leading to the proper folding of the
-lytic protease.
Similar conclusions have been reached following mutational studies of the subtilisin BPN' prodomain (Wang et al., 1998; Ruan et al., 1999
). Three mutations were engineered in the prodomain region, with none in contact with the subtilisin itself. By sequentially introducing these stabilizing mutations in the prodomain, the equilibrium for folding of the prodomain shifted dramatically, from 97% unfolded to 65% folded. As the prodomain was stabilized, the folding reaction of the subtilisin became faster and distinctly biphasic. Furthermore, consistently, the recent exciting work of Shinde et al. (1997, 1999) has elegantly illustrated that a mutation that has been engineered in this 70-residue Pro fragment resulted in an alternately folded subtilisin molecule, which has `memorized' the altered proregion conformation. This finding again illustrates the importance of the conformation of Pro and that its folding constitutes the first step in the folding of the precursor.
Proteins folding with the assistance of Pro have been shown to be under kinetic control (Baker et al., 1992; Cunningham et al., 1999
). Sauter et al. (1998) provide a compelling argument, rationalizing this choice by evolution. They point out that since proteins utilizing Pro to fold function in protease-infested environments, the need to be resistant to proteases is particularly acute. High kinetic barriers solve their problem. During folding, Pro permits lowering of the barriers, leading to the folded conformation. However, following the removal of the catalyst, climbing the barrier presents a very difficult hurdle, sustaining the native folds. Hence, in this case, the folding reaction is practically irreversible.
Aside from this kinetic role of the Pro region, recently it was demonstrated that a propeptide also acts to enhance the thermodynamic stability of the mature protease. Chymotrypsinogen and proelastase 2 are the only pancreatic proteases with propeptides that remain attached to the active enzyme via a disulfide bridge. It is likely that these propeptides are functionally important in the active enzymes, as well as in the zymogens. Kardos et al. (1999) have investigated the role of the disulfide-linked propeptide in the chymotrypsin(ogen) by comparing the stabilities of the wild-type with those of mutant proteins which lack propeptideenzyme interactions both in their zymogen (chymotrypsinogen) and in their active (chymotrypsin) forms. The mutants exhibited a substantially increased sensitivity to heat denaturation and to guanidine hydrochloride unfolding and a faster loss of activity at extremes of pH relative to those of their wild-type counterparts. The guanidine hydrochloride denaturation experiments indicated that the covalently linked propeptide provides about 24 kJ/mol of free energy of extra stabilization (G). Experimental evidence has further suggested that the propeptide of chymotrypsin restricts the relative mobility between the two domains of the molecule (Kardos et al., 1999
).
The important case of the chymotrypsinogen demonstrates that the propeptide can have a thermodynamic role and, at the same time, need not be cleaved off. Thus, it is reasonable to argue that it is possible that there exists an uncleaved, `hidden' intramolecular chaperone-like building block in some proteins. Unlike in the case of the proregion, in our case here of the uncleaved intramolecular chaperone-like catalyzed folding, the reaction is likely to be largely controlled by thermodynamics. This is the most frequently observed situation in proteins. We will formulate the concept and support our proposal with dihydrofolate reductase (DHFR).
All proteases have proregions: some fulfil both functional and intramolecular chaperone roles and others, like the zymogens, do not catalyze the folding reaction. From the evolutionary standpoint, these two types of cases are similar: both are synthesized as a single sequence, with the proregions linked to the enzymes and both have the same, inhibitory, functional role. However, for some enzymes belonging to the first type, nature has taken advantage of the proregions already being there, to assist in the folding, to reach a kinetically, otherwise inaccessible state. Hence there is an inherent evolutionary difference between the proregion intramolecular chaperones and molecules whose sole role is to catalyze folding, like the chaperonins and the small-molecule chaperones. Such molecules have a priori arisen for their explicit role in folding. Evolutionarily, the uncleaved intramolecular chaperone-like building block folding guide proposed here falls into the proregion category, namely, initially its role may have been designated for function. However, while already there, it may have evolved to assist optimally in the folding as well.
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The concept and mechanism of the intramolecular, uncleaved chaperone-like building block fragment |
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Our proposition is based on the building block folding model (Tsai et al., 1999a,b
). A building block is a contiguous sequence fragment, with a variable size. It is a highly populated, transient structural unit. The stability of a building block derives from its intra-fragment interactions. While the conformation of the building block that we observe in the native state of the protein is likely to be the one adopted by the fragment in solution, that is, it reflects the most populated conformation, the building block may also twist or open up and in so doing lose its intra-building block interactions. A building block constitutes a local minimum among all potential fragments. According to the building block folding model, via combinatorial assembly, the building blocks associate to form independently folding, compact, hydrophobic folding units. While the conformations of most building blocks are preserved in the folding process, the mutually stabilizing associations between the building blocks may still result in alternative conformations being selected in the combinatorial assembly process. The building blocks folding model is a `practical' model for protein folding, postulating that folding is a hierarchic process (Baldwin and Rose, 1999a
,b
). Here we propose that the amino-terminus which constitutes an intramolecular chaperone-like fragment is such a building block. It need not be stable and need not have a conformation which has a very high population time. However, it constitutes a local minimum among all of its neighbors and it has a higher population time than all other alternative conformations. The formation of such a building block during protein synthesis guards against misfolding, by preventing the mis-association of other building blocks. In the language of the energy landscapes and the folding funnels, a building block intramolecular chaperone acts by lowering the barrier of a misfolded conformation (Baker and Agard, 1994
; Gulukof and Wolynes, 1994
; Dill and Chan, 1997
; Karplus, 1997
). Hence an intramolecular building block which acts as a chaperone aids the trapped conformation climb out of its minima-well. By being chain-linked, this goal is achieved much more efficiently than otherwise.
Hence, specifically, here we argue that an intramolecular chaperone does not work via binding to any intermediate conformation of its adjoining building blocks and thereby inducing it to undergo a conformational change to the native conformation. On the contrary, here we propose that just as in intermolecular binding in general, the binding of the amino-terminal intramolecular chaperone to its adjoining building blocks is via conformational selection (Ma et al., 1999; Tsai et al., 1999c
; Kumar et al., 2000
). Among all the available conformations of the adjoining building blocks, those that bind are the ones whose binding produces the most favorable associations. And, in so doing, the equilibrium shifts in the direction of the native building block conformations. Such a proposition is consistent with the effect observed by Shinde et al. (1997). The mutation they introduced does not cause a direct, induced conformational change in the protease by `pushing' and `pulling' the protease into its `new native' state. Rather, the point mutation causes a change in the landscape, reflecting the effect of the altered conformation of the amino-terminal building block fragment. The mutant building block conformer preferentially selects different conformers from the populations of conformers of its adjoining building blocks, resulting in an altered subtilisin structure. However, after the proregion containing the mutation is cleaved, with time, we may expect the trapped altered structure to undergo a conformational change, overcoming the barriers, to the thermodynamically more stable native conformation.
For the sake of simplicity and clarity, here we outline the three major considerations in selecting a candidate intramolecular chaperone-like building block fragment. They are highlighted in the schematic illustration in Figure 1. First, the intramolecular chaperone fragment is most frequently at the amino-terminus of the protein; second, the candidate intramolecular chaperone-like building block mediates the interactions of other building blocks; and third, in its absence, non-native interactions between the building blocks are likely to take place. Figure 1a
depicts a protein (dihydrofolate reductase) cut into its three building block fragments. As the figure shows, in this case, the N-terminal fragment (in red) mediates the interactions of the second (green) and its sequentially connected third (blue) building block. Figure 1b
illustrates a hypothetical conformation which may arise if the N-terminal fragment is missing from the protein. Under such circumstances, a non-native association between the second and third building blocks may take place. This association is stable, as seen in Figure 1c
, resulting in a meta-stable state. Such a conformation has indeed been observed in simulations, in the absence of the N-terminal fragment (Y.Sham, B.Ma, C.-J.Tsai and R.Nussinov, unpublished results). Hence, as depicted in these figures, the intramolecular building block chaperone-like fragment is essential for achieving native interactions. Further, the more stable the building block, the faster will be the folding of the domain in which it resides.
Inspection of Figure 2a and b shows one likely reason for the role played by the proregion in the folding reaction. In both the
-lytic protease and subtilisin cases, the N-terminal building block segment mediates the interactions of other building blocks. It is essential that its conformation has a high population time. If the native conformation of this building block is present at a low concentration, the chances of non-native interactions between the other building blocks will be higher. Yet, inspection of Figure 2a
shows that the conformation of this building block fragment (in red), although constituting a local minimum, is still relatively unstable. On the other hand, its critical importance is seen from its mediating interactions with practically all other building blocks (in different colors). We may expect that a sequentially connected proregion would bind to this N-terminus fragment, since kinetically it is most favorable and thereby stabilizes it, enhancing the presence of this conformation. Consistently, a similarly unstable N-terminal (red) building block situation is observed in the
-lytic protease (Figure 2b
).
Hence, in the subtilisin (Wang et al., 1998), the mutations in the proregion shift the landscape of this domain, dramatically increasing the population of the conformation which is most favorable for binding with subtilisin and thereby driving the folding reaction. The higher the stability of a region which is in contact with other building blocks of the structure, the higher its population time is expected to be and the folding of the complex will also be faster. Wang et al. noted that under these conditions, the rate is determined by the isomerization of the trapped intermediate to the native complex structure. Consistent with the building blocks hierarchical model, the rate-limiting step here is the breaking of the non-native contacts between the building blocks, in the trapped mis-folded intermediate conformations. Hence, in both studies, the critical step is a stable and fast folding proregion, which binds, mediates and stabilizes less stable interactions between other building blocks in these structures and in this way drives the folding reaction.
Below we provide a concrete example showing this principle. We utilize our recently developed procedure for dissecting protein structures into building blocks, to produce progressively their anatomy trees, in terms of protein folding (Tsai et al., 2000).
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Does DHFR contain an intramolecular, uncleaved chaperone-like building block? |
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Additional potential candidates for uncleaved intramolecular chaperones |
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ADP-glucose pyrophosphorylase is a key regulatory enzyme in starch synthesis in most plant tissues. Laughlin et al. (1998) deleted small N- and C-terminal peptides on the large and, separately, on the small subunits from the potato protein and co-expressed these with their wild-type subunit counterparts from E.coli. Removal of the putative carboxy-terminal allosteric binding region from either subunit type prevents enzyme formation, indicating that the carboxy terminus of each subunit type is essential for proper subunit folding and/or enzyme assembly as well as its suggested role in allosteric regulation. Their results indicate that the N- and C-termini regions are essential for the proper catalytic and allosteric regulatory properties of the potato enzyme.
Recently, the L1 capsid protein of canine oral papillomavirus has been used as an effective systemic vaccine that prevents viral infections of the oral mucosa. The effect of this vaccine is critically dependent upon the native L1 conformation. Chen et al. (1998) generated a series of N- and C-terminal L1 deletion mutants. They found that (i) a deletion of the 26 C-terminal residues generated a mutant protein which folded correctly both in the cytoplasm and in the nucleus, (ii) further truncation of the L1 C terminus (67 amino acids) failed to express the conformational epitopes and (iii) deletion of the first 25 N-terminal amino acids also abolished expression of the conformational epitopes. However, the native conformation of this deletion mutant could be restored by the addition of the human papillomavirus type 11 N terminus. These results suggest that the L1 N-terminus has a critical role in protein folding.
The ß-subunit of Na,K-ATPase (ßNK) is an additional potential example. Complete truncation of the ßNK N-terminus allows for cell surface-expressed, functional Na,K-pumps that exhibit, however, reduced apparent K+ and Na+ affinities, as assessed by electrophysiological measurements. It has been shown that the ß N-terminus is not involved in the binding of K+ and Na+ and the -subunit. Rather, the effect of this domain is through the correct folding of the whole ß-subunit (Hasler et al., 1998
).
Most recently, Hayashi et al. (2000) studied the folding of ß-glucosidase. The enzyme is considered to be constructed from four regions: the N-terminal catalytic domain, the non-homologous region, the C-terminal unknown functional domain and C-terminal residues. Based on gene manipulations, these authors found that the folding information is unevenly distributed in these regions. The four types of truncated mutants at the non-homologous region resulted in active enzymes with slight modification of their characters. All four chimeric enzymes shuffled at the C-terminal unknown functional domain formed active enzymes. However, the two chimeric enzymes shuffled at the N-terminal catalytic domain were obtained as an inclusion body even though they were expressed with GroEL/ES. Refolding of these proteins by slow dialysis was not successful.
The above provide examples of either the N-terminal or other segments in the protein which play an important role in folding. These may constitute potential candidates for the uncleaved intramolecular chaperone-like building blocks. Careful examination of their structures will be needed to verify (or refute) them.
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The separate chaperones and the uncleaved intramolecular chaperone-like fragments |
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Sauter et al. (1998) have recently reported the structure of the -lytic protease complexed with the non-covalently bound proregion (Pro). The structure shows that a C-shaped Pro surrounds the C-terminal ß-barrel domain of the protease. However, the binding interface is arranged in such a way that the C-terminus of Pro is not adjacent to the N-terminus of the
-lytic protease. It therefore appears that when Pro is chain-linked to the enzyme, the binding orientation of
-lytic protease and Pro may differ from the most favorable conformation when the two molecular parts are unlinked. It is conceivable that when chain-linked, Pro may bind to its sequentially connected building block neighbor, forming a seed to which adjacent building blocks bind. In particular, it is noteworthy that when unbound, Pro is stable, with its conformation similar to that observed in the complex (Sauter et al., 1998
). That, however, is not the case for subtilisin, where the cleaved Pro is unstable (Wang et al., 1998
). On the other hand, in both
-lytic protease and the subtilisin cases, Pro is likely to aid in forming the active site regions, positioning itself in a way so as to enable the subsequent autocatalylic self-cleavage reaction.
Hence, taken together, this suggests that first, the cleaved, complexed structure is likely to differ to some extent from the structure of the chain-linked, prior to the cleavage event; Second, the folding pathways of the chain-linked molecule and of the protease without the Pro differ, as implied by the cutting and rearrangement observed in the complexed structure. Furthermore, in both the -lytic protease and the subtilisin cases, Pro serves as a template, to which adjacent building blocks bind, via selection of their most favorable conformers. Yet, after cutting, we observe a difference in the stability of Pro in the
-lytic protease as compared with that of subtilisin. In the subtilisin, Pro is stabilized through binding to its adjacent building blocks in the enzyme. Thus, while it is unstable, nevertheless the population of the native conformation of the Pro region of subtilisin is higher than that of all alternative conformations. This is consistent with the observations of Wang et al. cited above, namely that the subtilisin Pro is 95% unfolded. However, following the mutations they have introduced, Pro was observed to be 65% folded, with a marked increase in the folding rate, explained by the higher population time of Pro. Hence, the more stable the proregion, the faster is the rate of folding.
-Lytic protease, with a stable Pro, should fold faster than subtilisin. A similar observation has recently been made for the dominant role of the prosegment of prorenin in determining the rate of activation. Human prorenin activation by acid or by trypsin is faster than that of rat prorenin by two orders of magnitude. In an elegant experiment, two chimeric mutant prorenins were produced. The rate of activation of the first chimera, hPro/rRen, composed of human prorenin and the rat active renin segment, was as fast as the wild-type human prorenin. On the other hand, the rate of activation of the second chimera, rPro/hRen, composed of the rat prorenin prosegment and the human active renin segment, was as slow as that of the wild-type rat prorenin. These results suggest that the rate of activation of prorenin is predominantly determined by the N-terminal prosequence (Suzuki et al., 2000
).
Consistently, in the case of the uncleaved intramolecular chaperone-like proposed here, we may expect that the more stable the building block fragment acting as the intramolecular chaperone, the faster will be the rate of folding. The higher the stability, the larger is the population of the building block serving as a template for the binding of other building blocks of the structure. A similar behavior has already been observed in `simpler', two-state proteins. Folding of acylphosphatase is accelerated by the stabilization of local secondary structure elements (Chiti et al., 1999), although these do not play the role of intramolecular chaperones. Hence, going back to the dihydrofolate reductase example, we may predict that if the (red) N-terminal fragment is stabilized, e.g. via mutations, the rate of folding will increase. This prediction may be tested experimentally.
As for the kinetic control of protein folding, there are additional cases which fold without a Pro region and their folding still appears to be controlled by kinetics. Lomize et al. (1999) have simulated the folding of four proteins, adhering to three criteria: burial of non-polar side chains, saturation of `hydrogen-bonding potential' and stereochemical quality, as reflected in close packing with no hindrance or holes. They observed that some of the models they obtained had fewer exposed non-polar side chains, more hydrogen bonds and smaller holes than the corresponding crystal structures. This implies that the native structures of such proteins are under kinetic rather than thermodynamic control. In kinetically controlled folding reactions, with time, eventually the conformations will flip to their most stable state. However, the barriers are such that the time-scales may not be biologically relevant.
To date, it has been shown that folding pathways can be inferred from native structures (Alm and Baker, 1999a,b
; Galzitskaya and Finkelstein, 1999
; Munioz and Eaton, 1999
; Tsai et al., 2000
). An intriguing problem is how to infer which pathways are thermodynamically and which are kinetically controlled. Being able to distinguish between the two would constitute an important advance in the protein folding problem. To be very stable, a protein has a choice of two options: either to have a substantial energy gap between the native and the non-native conformations or to be surrounded by significant barriers. Hence a related question is an understanding of the general merit of kinetic versus thermodynamic control, where applicable.
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Some predictions |
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Conclusions
There are four major differences between the intermolecular and intramolecular proregion chaperones. First, while the intermolecular chaperones are sequence non-specific, the proregion is relatively sequence specific, accepting only a certain range of mutations. Still, even then, the mutations which change their own structure affect the conformation or the folding rate of the protease whose folding they catalyze. Second, the catalytic mechanism is different: while both work by lowering the barrier heights, the intermolecular chaperones or at least the chaperonins catalyze the folding reaction by unfolding misfolded conformations. On the other hand, the proregion catalyzes the reaction by directly assisting in the folding, through binding and stabilizing adjacent native building block conformations and thereby increasing their populations. Hence, macroscopically, in both intermolecular and intramolecular proregion chaperones, the funnel energy lanscape is similar; However, the microscopics differ. Third, there is a difference in the efficiency of the inter- as compared with the intramolecular chaperones. As the latter are chain-linked, their effective local concentration is higher. Fourth, in the case of the proregion, the corresponding proteins which fold with their assistance are under kinetic rather than thermodynamic control, unlike the intermolecular chaperone-assisted folding reactions. In addition, the two differ in their evolutionary aspect: whereas the proregions have primarily evolved to act as inhibitors, as indicated from their zymogen counterparts and have also assumed their folding role, the sole role of the intermolecular chaperones is to assist in the folding.
Here we propose that there are intramolecular chaperone-like building block fragments which fulfil the chaperone role and are not cleaved. The concept of intramolecular chaperones applies to a broad range of cases, where these fragments both assist in the folding reactions and, in addition, play a role in protein function. Hence, like the proregions, they fulfil a dual role. However, unlike the proregions, as their function is not to inhibit, they are uncleaved. Therefore, here we propose that cleavage of intramolecular chaperones is function-dependent. Mechanistically, the uncleaved intramolecular chaperone-like cases resemble the proregion-catalyzed reactions, lowering the barrier heights by selectively binding to the most favorable adjacent building block conformations. In this way, the equilbrium in the solution shifts toward these conformations, further driving the folding reaction (Tsai et al., 1999b; Kumar et al., 2000
). These intramolecular chaperone-like folding guides fulfil all the hallmarks of the intramolecular proregion-catalyzed reactions, with two differences: First, they remain covalently linked to their parent molecule after the native state has been reached; second, consequently, like the intermolecular chaperones, the folding reactions that they assist are controlled by thermodynamics. We suggest that this type of uncleaved intramolecular chaperone-like catalyzed folding reactions are a frequent occurrence. However, to preserve the definition of the chaperones, they are termed chaperone-like.
The mechanism proposed here is entirely different from the concept of `folding nuclei'. The concept of folding nuclei focuses on a non-sequential distribution of the folding information which controls the initial step in the folding process along the whole protein chain. On the other hand, our chaperone-like building block fragments proposition focuses on a segmental distribution of the folding information, which controls the population distribution throughout the hierarchical folding process.
Protein folding can be viewed as a process of intramolecular recognition. Hence, protein folding and proteinprotein association, which involves intermolecular recognition, are similar processes (Tsai et al., 1998, 1999b
). Both involve conformational selection, with subsequent population shifts in favor of the depleted conformer and thereby continue the binding/folding process. In both, the driving force is the hydrophobic effect, although charge and polar complementarity are also important contributors (Tsai et al., 1997
; Xu et al., 1997
). Recently, Eisenberg and co-workers demonstrated the implications of this concept for the detection of protein function and proteinprotein interactions directly from genome sequences (Marcotte et al., 1999
). Here we illustrate its implications for the molecular chaperones. The major difference is in the chain linkage and hence in the efficiency.
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Notes |
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Acknowledgments |
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References |
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Received May 15, 2000; accepted July 14, 2000.