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citric acid cycleimmediately raises questions of how and whether the crayfish are controlling the biomineralization; the excretion of these compounds, and perhaps key enzymes in their interconversion, represent a simple yet elegant physiological input on calcification. The question of whether there is an enzyme such as the mitochondrial pyruvate carboxylase present in the mineralizing space also arises. Pyruvate would then react with biotin-activated bicarbonate (HCO3) to form oxaloacetate, water and Pi. These could be the precursors for citrate, which Sato and colleagues6 found also associated with the ACC phase in vivo. The mechanism of the condensing enzyme would probably be similar to the one of citrate synthase (G = 32.2 kJ mol1), involving a citroyl-CoA thioester intermediate.

The broader picture of Sato and colleagues findings takes us back to the assumption that proteins and complex biological regulation would be needed for control over biomineralization. It is conceivable that Ca2+ and Mg2+ could distinctively interact with the resonance structures of phosphates and carboxylates present in organic metabolites such as PEP and citrate. The new results demonstrate that even small side groups (-CH3, -OH), or the delocalization potential of double bonds present in small molecules, could significantly impact the

respective phase equilibria of carbonate and phosphate minerals.

Storing energy in covalent bonds such as phosphoanhydrides is common to all organisms, leading back to the roots of life. With certain reservations, we can speculate what the ancient organic metabolism of energy-rich phosphates would have looked like, if an inorganic metabolism of H2O and CO2, HCO3, Ca2+ and CaCO3 acted as a temporary solidifying sink. This would certainly shift the chemical equilibrium of a particular series of chemical reactions. It could also protect the reactants from H2O, thus facilitating phosphorolysis rather than hydrolysis. This inspires speculation about whether ACC-Pi, ACC-PEP, ACC-3PG and calcite-citrate represent living fossils from the chemical history of life. An extremely hypothetical model (Fig. 1) outlines selected partial relationships between elementary biochemical conversions involving Pi, catalyzed today by GAPDH and pyruvate carboxylase, for example. Some of them could have their evolutionary origins in an ACC mineral shelter. Horowitz9 suggested that the origin of living matter by physicochemical means thus presupposes the existence of a highly complex chemical environment. With inorganic crystal growth in mind, Oparin10 proposed that the size and physicochemical structure

of a growing gel particle determines the capability of the smaller daughter particles to absorb and assimilate organic nutrients. This in turn selectively determines which particles (droplets) grow. With time, only particles with a certain history survive. Maybe the chemical compounds of Oparins first metabolizing gel were simple. If they included ACC, their phase diagrams might have been complex. Billions of years later, the crayfish took advantage.

Ingrid M. Weiss is in the Department of Materials in Biology, Leibniz Institute for New Materials, Saarbruecken, Germany. e-mail: ingrid.weiss@inm-gmbh.de

References1. Lowenstam, H.A. Science 211, 11261131 (1981). 2. Addadi, L., Raz, S. & Weiner, S. Adv. Mater. 15, 959970 (2003). 3. Beniash, E. et al. Proc. R. Soc. Lond. B 264, 461465 (1997). 4. Weiner, S. J. Struct. Biol. 163, 229234 (2008).5. Radha, A.V., Forbes, T.Z., Killian, C.E., Gilbert, P.U. &

Navrotsky, A. Proc. Natl. Acad. Sci. USA 107, 1643816443 (2010).6. Sato, A. et al. Nat. Chem. Biol. 7, 197199 (2011).7. Lippmann, F. Sedimentary Carbonate Minerals (Springer,

Heidelberg, 1973).8. Cornish-Bowden, A. (ed.). New Beer in an Old Bottle:

Eduard Buchner and the Growth of Biochemical Knowledge (Universitat de Valncia, Valncia, Spain, 1997).

9. Horowitz, N.H. Proc. Natl. Acad. Sci. USA 31, 153157 (1945). 10. Oparin, A.I. The Origin of Life on Earth. Trans. Ann Synge

(Academic, New York, 1957).

Competing financial interestsThe author declares no competing financial interests.

Intrinsically disordered proteins (IDPs) perform essential functions in organisms from all phyla. They are highly dynamic, do not form tertiary structures and contain variable amounts of transient secondary structure17. Many IDPs will fold into ordered structures when they bind to other proteins. This coupling of folding and binding is thought to balance the affinity and specificity of the interaction, but it remains unclear whether conformational flexibility is directly responsible for the ability of IDPs to specifically interact with different protein binding partners. In this issue, Kriwacki and colleagues demonstrate that the conformational flexibility of an intrinsically disordered inhibitor of cyclin-dependent kinases (Cdk)8,9, p21, is essential for binding and inhibiting different Cdks and their

associated cyclins. This work provides a clear example of how the conformational flexibility of an IDP can influence the binding to multiple protein partners.

In the Kriwacki study, nuclear magnetic resonance (NMR) spectroscopy was used to investigate the structure and dynamics of the kinase inhibitory domain (KID) of p21. This 61-residue, N-terminal domain of p21 contains three subdomains referred to as D1, LH and D2 (Fig. 1). In the ternary complex between p21-KID and Cdk2cyclin A, the D1 and D2 subdomains become ordered and interact directly with cyclin A and Cdk2, respectively. The LH subdomain adopts a helical structure that remains dynamic in the ternary complex. The LH subdomain tethers the D1 and D2 subdomains and has no direct role in the

binding interaction. To determine whether conformational flexibility influences the ability of p21-KID to bind and inhibit multiple Cdkcyclin complexes, the LH subdomain was lengthened (LH+3) or shortened (LH3) by three amino acids. Biophysical, biochemical and cell-based studies were performed to investigate the changes in structure, dynamics and function induced by these mutations.

Using circular dichroism, the authors showed that wild-type p21-KID increases the thermal stability of the Cdk2cyclin A complex by 20 C. This is presumed to happen because p21-KID is simultaneously binding to Cdk2 and cyclin A, promoting their association. In the p21-KID-LH+3 mutant, the thermal stability of the ternary complex is increased by another 3 C. In the

PROTEIN DYNAMICS

Bridging the gapDetailed biophysical and biochemical studies provide an exquisite example of how conformational flexibility controls the interaction between an intrinsically disordered protein and its numerous binding partners.

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wild-type complex, the LH subdomain is stretched to the point where one of the helical turns forms a noncanoncial structure (Fig. 1). The increase in thermal stability observed for the p21-KID-LH+3 mutant is probably due to some combination of relaxing the stretched helix and optimizing the position of the D1 and D2 subdomains for binding cyclin A and Cdk2, respectively. This latter idea is supported by the fact that the p21-KID-LH+3 mutant has a fourfold lower half-maximal inhibitory concentration for Cdk2cyclin A inhibition when compared to wild type. Shortening the LH subdomain by three amino acids has a much more dramatic effect on ternary complex formation. In the p21-KID-LH3 mutant, thermal stability of the ternary complex is reduced by 12 C relative to wild type. This mutant also has a striking effect on function, increasing

the half-maximal inhibitory concentration for Cdk2cyclin A inhibition by forty-fold relative to wild type. Interestingly, different inhibitory effects were observed with different combinations of Cdkcyclins, and these effects were generally consistent with the different distances that the LH subdomain must span in the ternary complex.

The work by Kriwacki and colleagues provides a detailed mechanistic interpretation of how the inherent flexibility of an IDP can result in structural adaptability and functional promiscuity. Further studies are required to determine how this structural adaptability is manifested during the binding process. It is expected that the amount of conformational flexibility in the free state of an IDP will play a major role in determining the binding mechanism. The study by Kriwacki and colleagues shows that the amount of

conformational flexibility in the bound state must also be taken into consideration to comprehensively describe the binding mechanisms of IDPs. It is clear from this study, and from the work of others in this field, that the conformational flexibility of IDPs has evolved to an exquisite level where the motion of two or three amino acids can have a major effect on function. In hindsight, it is obvious that the conformational flexibility of IDPs provides a rich framework for evolutionary selection, and it is apparent that the future of structural biology depends greatly on our ability to precisely define how this flexibility influences function.

Gary W. Daughdrill is in the Department of Cell Biology, Microbiology and Molecular Biology and the Center for Drug Discovery and Innovation, University of South Florida, Tampa, Florida, USA. e-mail: gdaughdrill@usf.edu

References1. Eliezer, D. Curr. Opin. Struct. Biol. 19, 2330 (2009).2. Dyson, H.J. & Wright, P.E. Nat. Rev. Mol. Cell Biol. 6, 197208

(2005).3. Wright, P.E. & Dyson, H.J. Curr. Opin. Struct. Biol. 19, 3138 (2009).4. Dunker, A.K. et al. BMC Genomics 9 Suppl 2, S1 (2008).5. Mittag, T. & Forman-Kay, J.D. Curr. Opin. Struct. Biol. 17, 314

(2007).6. Tompa, P. Structure and Function of Intrinsically Disordered

Proteins, 331 (Taylor and Francis Group, Boca Raton, 2010).7. Daughdrill, G.W., Pielak, G.J., Uversky, V.N., Cortese, M.S. &

Dunker, A.K. Natively disordered proteins. in Protein Folding Handbook, Vol. 3 (eds. Buchner, J. & Kiefhaber, T.) 275357 (Wiley-VCH, Darmstadt, 2005).

8. Xiong, Y. et al. Nature 366, 701704 (1993).9. Wang, Y. et al. Nat. Chem. Biol. 7, 214221 (2011).10. Russo, A.A., Jeffrey, P.D., Patten, A.K., Massague, J. & Pavletich, N.P.

Nature 382, 325331 (1996).

Competing financial interestsThe author declares no competing financial interests.

KACRRLFGPVDSEQLSRDCDALMAGCIQEARERWNFDFVTETPLEGDFAWERVRGLGLPKLYLPD1 LH D2

Shorten the helix bythree residues (LH3)

Lengthen the helix bythree residues (LH+3)

Wild-type LHsubdomain Stabilizes Cdk2cyclin A complex by 20 C

Stabilizes Cdk2cyclin A complex by 23 C

Stabilizes Cdk2cyclin A complex by 8 C

EQLSRDCDALMAGCIQEARERALR

EQLSRDCDALMAGCIQEA

Figure 1 | Schematic showing the subdomains of the kinase inhibitory domain of p21. The D1 subdomain forms an extended structure when bound to cyclin, the D2 subdomain forms extended, sheet and turn structures when bound to Cdk and the LH subdomain forms a dynamic, helical structure in the ternary complex. Red text shows the amino acid sequence for the wild-type LH subdomain as well as the LH+3 and LH3 mutants. A ribbon diagram is shown for the wild-type LH subdomain (figure based on the p27Cdk2cyclin A crystal structure; PDB ID: 1JSU10). Approximate changes in the length of the LH+3 and LH3 mutant subdomains are also shown.

The nematode C. elegans is frequently used as an animal model to study the genetics of fat storage and metabolism13. Because these processes are conserved between worms and mammals, C. elegans is convenient as well as genetically tractable. For example, the human Bardet-Biedl syndrome causes profound obesity; knockdown of worm orthologs of genes mutated in this disorder similarly cause excessive fat storage2. Thus, findings

made by genetic screening in worms have a good chance of being relevant in higher systems. Although whole- organism screens allow researchers to probe more complex biology than cell- based or biochemical assays4, with a more nuanced analysis of cell-type interactions, enabling technologies have only recently been developed to perform screens efficiently in C. elegans5,6 or zebrafish7,8. Lemieux et al.9 have extended these

efforts to small molecules and report a high-throughput screen for fat storage in C. elegans, identifying a novel agonist of AMP-activated kinase (AMPK) capable of reducing fat storage.

Disruption of energy homeostasis can lead to disease. Although too much fat in humans causes obesity and leads to increased risks of heart disease and type 2 diabetes, too little fat is a problem as well. Lipodystrophy causes increased plasma triglycerides and is

SCREENING

Low-fat worms on drugsHigh-throughput screening in Caenorhabditis elegans identified a compound that distinctly regulates fat storage and feeding, highlighting new players in energy homeostasis.

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