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Allostery in the Ferredoxin Protein Motif Does Not Involve a Conformational Switch

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Allostery in the Ferredoxin Protein Motif Does Not Involve a Conformational Switch Allostery in the ferredoxin protein motif does not involve a conformational switch Rachel Nechushtaia, Heiko Lammertb, Dorit Michaelia, Yael Eisenberg-Domovicha, John A. Zurisc, Maria A. Lucac, Dominique T. Capraroc, Alex Fisha, Odelia Shimshona, Melinda Royc, Alexander Schugb,d, Paul C. Whitforde, Oded Livnaha, José N. Onuchicb,1, and Patricia A. Jenningsc,1 aLife Science Institute and The Wolfson Centre for Applied Structural Biology, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem 91904, Israel; bCenter for Theoretical Biological Physics and the Department of Physics, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0375; cDepartment of Chemistry and Biochemistry, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093- 0375; dDepartment of Chemistry, Umeå University, Umeå, Sweden; and eLos Alamos National Laboratory, Theoretical Biology and Biophysics, MS K710, Los Alamos, NM 87545 Contributed by José N. Onuchic, December 28, 2010 (sent for review December 11, 2010) Regulation of protein function via cracking, or local unfolding substantial theoretical (9–13) and experimental (14, 15) work and refolding of substructures, is becoming a widely recognized has aimed at revealing the details of the functionally relevant mechanism of functional control. Oftentimes, cracking events are transition-state ensembles. These investigations have shown localized to secondary and tertiary structure interactions between that rearrangements may involve a degree of cracking, or loca- domains that control the optimal position for catalysis and/or the lized unfolding and refolding, which serves to reduce the asso- formation of protein complexes. Small changes in free energy ciated free-energy barriers (16, 17). By modulating the balance associated with ligand binding, phosphorylation, etc., can tip the of these order–disorder transitions that control domain–domain balance and provide a regulatory functional switch. However, interactions/orientations, cells may tune the functional dynamics. understanding the factors controlling function in single-domain Despite these conceptual advances in understanding enzyme reg- proteins is still a significant challenge to structural biologists. We ulation, these principles may not be applicable to single-domain investigated the functional landscape of a single-domain plant- proteins if they do not exhibit large-scale structural rearrange- type ferredoxin protein and the effect of a distal loop on the elec- ments. In those cases, alternative modes of functional control BIOPHYSICS AND tron-transfer center. We find the global stability and structure are are likely employed by the cell. COMPUTATIONAL BIOLOGY minimally perturbed with mutation, whereas the functional prop- In the present study, we investigate the relationship between erties are altered. Specifically, truncating the L1,2 loop does not structure, dynamics, and function for a single-domain plant-type lead to large-scale changes in the structure, determined via X-ray ferredoxin (Fdx) protein. This Fdx folding motif is among the crystallography. Further, the overall thermal stability of the protein most abundant in nature, and it has been well studied previously is only marginally perturbed by the mutation. However, even biochemically (18–23), making Fdx an excellent candidate for though the mutation is distal to the iron–sulfur cluster (∼20 Å), it quantitative, biophysical characterization. Moreover, the plant- leads to a significant change in the redox potential of the iron– type Fdx folding motif has been found in numerous redox pro- sulfur cluster (57 mV). Structure-based all-atom simulations indi- teins and enzymes, as well as in functionally unrelated proteins cate correlated dynamical changes between the surface-exposed that do not contain the [2Fe-2S] cluster, such as ubiquitin and loop and the iron–sulfur cluster-binding region. Our results suggest the immunoglobulin-binding domain of protein G (24, 25). Cir- intrinsic communication channels within the ferredoxin fold, com- cular permutation of the secondary structure units helped identify posed of many short-range interactions, lead to the propagation of previously unrecognized homologs, such as the mercury transpor- long-range signals. Accordingly, protein interface interactions that ter protein MerP (26). This versatility suggests that a fundamen- ’ involve L1,2 could potentially signal functional changes in distal tal understanding of the fold s dynamical properties may facilitate regions, similar to what is observed in other allosteric systems. the design of proteins with tightly regulated, and unique, func- tional roles (27). The development of “modular” proteins that electron transfer ∣ functional energy landscape ∣ iron–sulfur proteins ∣ can be artificially assembled into large functional complexes is protein folding also at the heart of modern synthetic biology (28). When com- plexed with iron–sulfur clusters, Fdx proteins possess electron ver the last several decades, our understanding of protein transport properties, which suggests they could be a component function has evolved from a rather static perspective, where in artificial photosynthetic devices and other biologically moti- O vated molecular “circuitry.” signaling and function have been understood through surface The potential role of iron–sulfur (FeS) proteins in synthetic complementarity arguments, such as the “lock and key” paradigm biology is inspired by their role inside the cell. These proteins (1, 2), to a more dynamic view where protein conformational are key players in the three life-essential processes: photosynth- fluctuations are inextricably linked to function (3). As our under- esis, nitrogen fixation, and respiration (29, 30). The plant-type standing of protein dynamics expands, we are revealing many Fdx proteins carry a single surface-exposed [2Fe-2S] iron–sulfur mechanisms by which proteins exploit conformational fluctua- tions to perform cellular function. In multidomain proteins, relative repositioning of domains is often linked to their levels Author contributions: R.N., H.L., D.M., Y.E.-D., J.Z., M.A.L., D.T.C., A.F., O.S., M.R., A.S., of activity. For example, large-scale domain rearrangements in P.C.W., O.L., J.N.O., and P.A.J. designed research; R.N., H.L., D.M., Y.E.-D., J.Z., M.A.L., D.T.C., A.F., O.S., M.R., and A.S. performed research; R.N., H.L., P.C.W., O.L., and P.A.J. the four-domain Src kinase (4) and C-terminal Src kinase (5, 6) analyzed data; and R.N., H.L., P.C.W., O.L., J.N.O., and P.A.J. wrote the paper. lead to these proteins being in so-called “on” or “off” states, and The authors declare no conflict of interest. functional regulation may be obtained by adjusting the balance Data deposition: The crystallography, atomic coordinates, and structure factors have been between these conformations (7). In proteins such as adenylate deposited in the Protein Data Bank, www.pdb.org (PDB ID code 3P63). kinase, domain rearrangements can be rate limiting during each 1To whom correspondence may be addressed. E-mail: [email protected] or round of catalysis (8), where the enzyme cycles between ligand- [email protected]. competent and ligand-release conformations. Because the kinet- This article contains supporting information online at www.pnas.org/lookup/suppl/ ics of these transitions control the activity level of the protein, doi:10.1073/pnas.1019502108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1019502108 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 30, 2021 cluster (ISC) (19), and homologous proteins are found in all organisms, e.g., adrenodoxin in human mitochondria (20). The E oxidation-reduction potentials ( m) of these Fdx proteins range C’ from −325 mV to around −450 mV (21), making Fdx one of the strongest soluble reducing agents in nature (22). The plant-type Fdx functions primarily as the electron acceptor of photosystem I, and upon reduction Fdx functions as an electron donor in a myr- iad of key metabolic pathways (e.g., NADPþ reduction, carbon assimilation, nitrite and sulfite reduction, glutamate synthesis, and thioredoxin reduction, etc.) (22, 23, 31–35). The Fdx fold (22, 23, 31, 36), also termed the β-grasp, or UB roll, is found throughout the cell. The minimum core elements N’ consist of a four-stranded mixed β-sheet with an α-helix packed across one face. Loop insertions and additional strands/helices may be incorporated into this fold for functional purposes. A rib- bon diagram of the tertiary fold of Mastigocladus laminosus Fdx (mFdx), is shown in Fig. 1 (19, 37). Substitution of the flexible L1,2 loop (residues 10–14) of wild-type Fdx (mFdxWT) with the rigid β-turn of a mesophilic Fdx (residues 10–12) (here we refer to this substituted mutant as mFdxΔL1;2),* yields a protein that is functional at 23 °C but loses electron-transfer (ET) activity at 55 °C (19), whereas mFdx is fully active. This loss of function is not due to dissociation of the [2Fe-2S] cluster, as this moiety is stable to 75 °C. The highly spe- L1,2 cific effects of this isolated mutation is a well-defined feature, β1 making mFdx ideal for exploring the relationship between a pro- tein’s functional and dynamical properties. Through a combined experimental-theoretical investigation, we find that the network of short-range residue–residue interac- tions that define the Fdx fold directly facilitates long-range communication between L1,2 and the ISC pocket. Protein–film voltammetry (PFV) showed that the deletion of loop L1,2 alters the redox potential of the functional [2Fe-2S] center, ca. 20 Å re- moved from the site of mutation. X-ray crystallography and tem- perature-dependent CD studies revealed that, despite changes in β2 the ET rates at high temperatures, the global structure and stabi- lity of the protein are robust. The only noticeable structural Fig. 1. Loop deletion does not have global effects on the structure of mFdx. changes (i.e., larger than the uncertainties in the crystallographic (Top) Ribbon representation of M.
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