Allostery in the ferredoxin protein motif does not involve a conformational switch

Abstract

Regulation of protein function via cracking, or local unfolding and refolding of substructures, is becoming a widely recognized mechanism of functional control. Oftentimes, cracking events are localized to secondary and tertiary structure interactions between domains that control the optimal position for catalysis and/or the formation of protein complexes. Small changes in free energy associated with ligand binding, phosphorylation, etc., can tip the balance and provide a regulatory functional switch. However, understanding the factors controlling function in single-domain proteins is still a significant challenge to structural biologists. We investigated the functional landscape of a single-domain planttype ferredoxin protein and the effect of a distal loop on the electron-transfer center. We find the global stability and structure are minimally perturbed with mutation, whereas the functional properties are altered. Specifically, truncating the L1,2 loop does not lead to large-scale changes in the structure, determined via X-ray crystallography. Further, the overall thermal stability of the protein is only marginally perturbed by the mutation. However, even though the mutation is distal to the iron-sulfur cluster (∼20 Å), it leads to a significant change in the redox potential of the ironsulfur cluster (57 mV). Structure-based all-atom simulations indicate correlated dynamical changes between the surface-exposed loop and the iron-sulfur cluster-binding region. Our results suggest intrinsic communication channels within the ferredoxin fold, composed of many short-range interactions, lead to the propagation of long-range signals. Accordingly, protein interface interactions that involve L1,2 could potentially signal functional changes in distal regions, similar to what is observed in other allosteric systems.electron transfer | functional energy landscape | iron-sulfur proteins | protein folding O ver the last several decades, our understanding of protein function has evolved from a rather static perspective, where signaling and function have been understood through surface complementarity arguments, such as the "lock and key" paradigm (1, 2), to a more dynamic view where protein conformational fluctuations are inextricably linked to function (3). As our understanding of protein dynamics expands, we are revealing many mechanisms by which proteins exploit conformational fluctuations to perform cellular function. In multidomain proteins, relative repositioning of domains is often linked to their levels of activity. For example, large-scale domain rearrangements in the four-domain Src kinase (4) and C-terminal Src kinase (5, 6) lead to these proteins being in so-called "on" or "off" states, and functional regulation may be obtained by adjusting the balance between these conformations (7). In proteins such as adenylate kinase, domain rearrangements can be rate limiting during each round of catalysis (8), where the enzyme cycles between ligandcompetent and ligand-release conformations. Because the kinetics of these tra...

Allosteric control in a metalloprotein dramatically alters function

“…This long-range allosteric control of the metal center suggests that many other MPs may in fact require full use of their uniquely evolved scaffolds to perform complex biological tasks. Taken together, this work provides a foundation for investigating how metal centers in metalloproteins are influenced by the global motions and expands our understanding beyond the control of simple electron transfer by distal mutations (43). This approach is critical for designing new therapeutics for targeting this class of [2Fe-2S] proteins, as well as de novo design of new metalloproteins (44,45).…”

Two-step Mechanism for Modifier of Transcription 1 (Mot1) Enzyme-catalyzed Displacement of TATA-binding Protein (TBP) from DNA

“…Alternatively, DNA unbending could be a consequence of a conformational change in TBP itself induced by Mot1 binding (36). As the Mot1 NTD-TBP co-crystal does not reveal any significant change in TBP structure compared with TBP alone (35), such an effect on the DNA binding activity of TBP apparently would occur via a more subtle allosteric mechanism (14,(62)(63)(64)(65).…”

Simulating Biomolecular Folding and Function by Native‐Structure‐Based/Go‐Type Models

“…[102,106,115,116] Recent work using SBM investigated the effect of symmetry on the uniqueness of native states for several proteins, [113,[117][118][119] the folding of complex topologies such as knotted proteins, [120][121][122] the binding of Zn-finger proteins to DNA, [123] or proteinÀprotein association. [124][125][126][127] As an example, the complex topology of protein knots is conserved within several protein families and is assumed to have the important physiological role of additional fold stabilization. Different knotted proteins are identified and classified on the basis of their knot types.…”

“…Understanding the detailed mechanism of how the short-range conformational changes of the distal loops could influence the function of the protein at the regulator site would be helpful in the design of potent therapeutic agents, and therefore, attracted major research interest. The coupling of the folding mechanism and allosteric properties of two metalloproteins, mito-NEET [130][131][132] and ferredoxin, [124] were examined using SBM.…”