PERSPECTIVE

Whence cometh the allosterome?

Janet E. Lindsley† and Jared Rutter† Department of , University of Utah, 15 North Medical Drive East, Room 4100, Salt Lake City, UT 84112-5650

Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved June 5, 2006 (received for review May 31, 2006)

Regulation of cellular functions can be accomplished by many mechanisms, including transcriptional regulation, alternative splicing, translational regulation, and other posttranslational covalent modifications, degradation, localization, – protein interactions, and small-molecule allosteric effectors. Largely because of advances in the techniques of molecular biology in the past few decades, our knowledge of regulation by most of these mechanisms has expanded enormously. Regulation by small- molecule, allosteric interactions is an exception. Many of the best-known allosteric regulators were discovered decades ago, when we knew little about all of these other forms of regulation. Allostery is the most direct, rapid, and efficient regulatory mechanism to sense changes in the concentration of small molecules and alter cellular responses to maintain homeostasis. In this perspective, we present the argument that is underappreciated in the systems biology world and that many allosteric effectors remain to be discovered.

wo of the earliest examples of cules. It can be very rapid, on the order past 4 decades, whereas those describing allosteric regulation are the of the rate of diffusion (Ϸ108 MϪ1⅐sϪ1), allosteric regulation have not (see Fig. control of binding to and readily reversible. And unlike 1). Although scientists have described by protons (1) and changes in covalent protein modification transcriptomes, proteomes, interactomes, TAMP activation of glycogen phosphory- such as phosphorylation͞dephosphoryla- and kinomes, the ‘‘allosterome’’ is still a lase (2). During the heyday of metabolic tion cycles, no cellular energy is used up mystery. research, allosteric regulators were ac- in allosteric signaling. Second, a rela- How are allosteric regulators generally tively sought out. By the time that the tively large percentage of the found? In the past, they were primarily ‘‘Central Dogma of Molecular Biology’’ studied 40 years ago were known to be found either by serendipity or metabolic was proposed (3), several examples of allosterically regulated, and, although intuition followed by experimental verifi- ‘‘end-product’’ or ‘‘’’ inhibition the number of known proteins has sky- cation. For example, the discovery of the were already known (4–7). In each case, rocketed, the number of known alloste- allosteric activation of the AMP-activated the end product of a metabolic pathway ric regulators has not. Forty years ago, protein (AMPK) was a combina- was shown to inhibit an early in the effort required to discover a new tion of these methods. Kinetic studies the pathway. Surprisingly, in each case protein was at least that required to dis- showed that this kinase had maximal ac- the inhibitor was ‘‘not a steric analogue cover a small-molecule regulator. Today, tivity at 400 ␮M ATP and barely detect- of the ,’’ and in 1961 Monod gene and protein discovery is systematic, able activity at 4 mM ATP (12). The and Jacob (8) coined the term ‘‘alloste- whereas small-molecule discov- protein substrate of AMPK that was being ric inhibition’’ to indicate inhibitory in- ery still occurs one protein at a time studied, acetyl CoA carboxylase, catalyzes teractions at a site distinct from the and typically only on the rare occasions the committed step in . This first discussion of allo- when the investigator has a reason to and is inhibited by phosphorylation. It steric was prescient in many look for such regulators. made sense that fatty acid synthesis respects; they proposed the following: The mechanisms of regulation that should be inhibited when the energy (i) that allostery need not be restricted have primarily been revealed in the last charge of the is low, providing the to end-product inhibition and could be a two decades are those that could be rationale for testing whether AMP general form of regulation, (ii) that it discovered by the tools of molecular activates the acetyl CoA carboxylase- might be very difficult to predict the biology. Our abilities to synthesize, ma- inhibiting kinase. A relatively high AMP͞ chemical nature of allosteric effectors nipulate, and amplify nucleic acids, as ATP ratio is now known to activate and therefore to discover them, and (iii) well as to create specific antibodies, AMPK, and this enzyme is often called that allosteric sites might be even more have made it possible to discover tran- the cellular ‘‘energy gauge’’ or ‘‘fuel useful as drug targets than active sites. scriptional and posttranscriptional sensor’’ (13). By 1965, there were at least 31 allosteric modes of regulation on a large scale. More recently, several allosteric effec- effectors known (9), but only 1 protein Transcriptional regulation can be stud- tors have been discovered from high- kinase͞substrate pair ( ied by using promoter fusion constructs, throughput chemical screens in search of kinase͞glycogen phosphorylase) had chromatin immunoprecipitation, mi- specific enzyme activators or inactiva- been identified (10, 11). croarrays, quantitative RT-PCR, North- tors. For example, metabolic studies How common is allosteric regulation? ern blots, etc. Likewise, the analysis of provided no reason to suspect that mo- Is it primarily restricted to the commit- cellular regulation by splicing, mRNA nomeric would be regulated ted step of metabolic pathways, or is it a stability, translation, phosphorylation, by allostery. However, it was known that widespread, perhaps even universal, , etc. has become possible increased glucokinase activity could de- phenomenon? The answers to these because of similar tools. In contrast, crease levels, and hence questions are not yet known, but there these tools have not yet resulted in there was a strong motivation to search are many reasons to think that allosteric widely used, routine methods to dis- regulation is not rare. First, allostery is cover small, allosteric effectors. One Conflict of interest statement: No conflicts declared. an ideal way for proteins (and ) to consequence is that the number of stud- This paper was submitted directly (Track II) to the PNAS sense the changing milieu of a cell and ies describing modes of regulation ame- office. respond to maintain cellular homeosta- nable to molecular biology techniques, †To whom correspondence may be addressed. E-mail: janet. sis. Allostery provides a mechanism to such as transcription or phosphorylation, [email protected] or [email protected]. directly sense the levels of small mole- have increased dramatically over the © 2006 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0604452103 PNAS ͉ July 11, 2006 ͉ vol. 103 ͉ no. 28 ͉ 10533–10535 Downloaded by guest on September 30, 2021 optimization of the initial compound (14). The unique capability of the disul- fide-tethering technique is the ability to find low-affinity effectors. Many of the small molecules in a cell that may be sensed by allosteric proteins are present at relatively high concentrations (micro- molar to millimolar). For a protein to sense the change in such a metabolite, the affinity would need to be low, and low-affinity interactions are difficult to discover. This novel covalent tethering method side-steps the need for high affinity. As illustrated by the two examples of newly discovered allosteric regulators described above, pharmacologists have realized the utility of searching for allo- steric effectors in addition to, or instead of, active-site ligands. Active-site ligands are constrained by the active-site chem- istry, whereas allosteric sites have no such constraints. Active-site structures are often quite similar between distinct proteins, presenting a challenge for drug Fig. 1. Percent of papers listed in PubMed (www.ncbi.nlm.nih.gov͞entrez͞query.fcgi) describing tran- selectivity. Allosteric sites are not gener- scription, phosphorylation, and allostery during the past 4 decades. Titles and abstracts were searched for ally conserved between different pro- the following terms: ‘‘alloster*,’’ ‘‘’’ OR ‘‘’’ OR ‘‘ser͞thr kinase’’ OR ‘‘phos- teins (19, 20). Although the idea that phorylase kinase’’ OR ‘‘phosphoprotein’’ OR ‘‘,’’ and ‘‘transcription*.’’ The num- drugs targeting allosteric sites might be ber of papers identified was divided by the total number of papers published during the time interval. even more effective than those targeting active sites has only recently become a popular notion among pharmacologists, for activators of this enzyme as potential Wells and colleagues (17–19). These it was proposed in the first paper defin- diabetes therapeutics. A high-throughput researchers have used a high-through- ing allostery (8). chemical screen revealed a class of put, disulfide-tethering technique to find In addition to the problem that allo- small-molecule activators that both in- small-molecule allosteric inhibitors of steric effectors are not generally easy to creases the kcat and decreases the S0.5 caspase-1, -3, and -7. For caspase-1 and identify, the relative lack of interest in for glucose and lowers the blood glucose -7, the allosteric sites are located at the discovering new allosteric regulators by Ϸ concentration in rodent models of dia- dimer interfaces, 15 Å from the active biologists may stem from a misconcep- betes (14). Crystal structures of the en- sites. Binding of the allosteric inhibitors tion about what types of proteins are zyme plus and minus the and stabilizes conformations of the caspases susceptible to allosteric regulation. Allo- glucose indicate that the activator binds that are catalytically inactive and are steric regulation simply means that a Ϸ 20 Å from the active site and appears similar to the zymogen forms of the protein’s activity is regulated by binding to stabilize an active conformation of ; in other words, the inhibitors of a at a site other than the ac- the enzyme (15). Five known activating functionally reverse zymogen activation tive site. By this definition, all proteins mutations of glucokinase, discovered (17, 19). The authors postulate the exis- could be allosterically regulated, an idea from patients with hypoglycemia, map tence of an endogenous allosteric inhibi- that has been suggested by others (21). to the same site (16). Additionally, a tor that would act as a safeguard against However, there is a common perception glucokinase mutant (V62M) discovered spurious caspase activation. that only oligomeric proteins, particu- in a patient with maturity-onset diabetes This high-throughput, disulfide- larly those with multiple identical sub- of the young, type 2, also resides at this tethering technique addresses two of units, can be allosterically regulated. site. The purified V26M glucokinase has the major difficulties in discovering This perception may stem from the his- a decreased S0.5 for glucose, the oppo- allosteric effectors: unknown chemical tory of the discovery of allosteric regu- site of what one would expect for a pa- composition and low affinity. Although lators. The Monod–Wyman–Changeux tient with hyperglycemia (and from the proteins are made from amino acids and (MWC) and Koshland–Ne´methy–Filmer other known MODY2 mutants) (16). nucleic acids are made from nucleosides, (KNF) models were proposed in the Intriguingly, the V26M mutant does not small-molecule effectors come from a 1960s to explain the allosteric regulation respond to stimulation by the pharmaco- wide spectrum of chemical classes. Al- seen in hemoglobin and several meta- logical allosteric activator (16). This though it is often possible to predict bolic regulatory enzymes (9, 22). Several combination of findings about the what compounds might bind at the ac- of the best studied of these systems not V26M mutant (less active in vivo, more tive site based on the enzymatic reaction only exhibited heterotrophic regulation active in vitro, and resistant to the phar- that occurs there, no such rules apply to (meaning regulation by small molecules macological allosteric activator) strongly allosteric sites. Therefore, an unbiased that were distinct from the substrates or suggests the existence of an endogenous screen of a diverse, small-molecule li- primary ligand) but also exhibited posi- allosteric regulator of glucokinase. brary is crucial. The glucokinase activa- tive or positive homotropic A second example of ‘‘where there’s a tor described above was initially found regulation. For example, oxygen is both will there’s a way’’ to discover allosteric in a screen of 120,000 structurally di- the primary ligand for hemoglobin and, regulators is the analysis of caspases by verse compounds, followed by chemical because it binds with positive cooperat-

10534 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0604452103 Lindsley and Rutter Downloaded by guest on September 30, 2021 ivity, is a positive homotropic regulator zymes consist of multiple subunits and meric AMP͞ATP sensing domain (13). of hemoglobin. It is this positive cooper- multiple active sites.‘‘ Additionally, a re- Therefore, although one’s favorite ativity or homotropic regulation that cent high-profile review of allosteric protein may not have an oligomeric causes the sigmoidal binding and initial mechanisms in signaling pathways focused structure similar to hemoglobin, phos- velocity curves so commonly associated on the WMC theory and hence only dis- phofructokinase, or aspartate transcar- with allosteric proteins. It is important cussed allostery as it applies to oligomers, bamoylase, it may still be regulated by to realize that the MWC and KNF mod- particularly homooligomers (24). allostery. els were developed as inclusive models Although many of the originally dis- In summary, even though recent tech- that could explain both the positive co- covered allosterically regulated proteins nological advances have not facilitated operativity and the heterotropic al- happened to be oligomers, there is no a systematic discovery of small-molecule, lostery seen in many of the proteins that priori reason why heterotropically regu- allosteric regulators, a great many may have been studied to date. Although lated proteins need to be multimers, well exist. It is difficult to imagine that these landmark papers have brilliantly particularly homooligomers. In fact, most of the biologically important allo- guided the subsequent studies on the there are many allosterically regulated steric regulators were already discovered allosteric regulation of oligomeric pro- monomeric and heterooligomeric pro- before the days of PCR and commer- teins, and particularly positive cooperat- teins that do not show cooperativity in cially available restriction enzymes; ivity, they also have been misinterpreted substrate binding or catalysis. Two ϩ there must be more just waiting to be by some to indicate that only oligomeric examples include Ca2 binding by recov- revealed. proteins can be allosterically regulated. erin (25, 26) and PAS-domain-contain- In fact, even a trusted textbook of bio- ing cytoplasmic histidine (27). We thank Drs. Timothy Formosa, Martin C. chemistry (23), when discussing alloste- The cellular fuel sensor AMPK, al- Rechsteiner, and Steven L. McKnight for ric enzymes, states that ‘‘[t]hese en- though not a monomer, has a mono- thoughtful critiques of this manuscript.

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