Global Conformational Rearrangements Research Articles

Electron Paramagnetic Resonance Spectroscopy of Site-directed Spin Labels Reveals the Structural Heterogeneity in the N-terminal Domain of ApoA-I in Solution

Apolipoprotein A-I (apoA-I) is the major protein constituent of high density lipoprotein (HDL) and plays a central role in phospholipid and cholesterol metabolism. This 243-residue long protein is remarkably flexible and assumes numerous lipid-dependent conformations. Consequently, definitive structural determination of lipid-free apoA-I in solution has been difficult. Using electron paramagnetic spectroscopy of site-directed spin labels in the N-terminal domain of apoA-I (residues 1-98) we have mapped a mixture of secondary structural elements, the composition of which is consistent with findings from other in-solution methods. Based on side chain mobility and their accessibility to polar and non-polar spin relaxers, the precise location of secondary elements for amino acids 14-98 was determined for both lipid-free and lipid-bound apoA-I. Based on intermolecular dipolar coupling at positions 26, 44, and 64, these secondary structural elements were arranged into a tertiary fold to generate a structural model for lipid-free apoA-I in solution.

Identification of Two Distinct Hybrid State Intermediates on the Ribosome

High spatial and time resolution single-molecule fluorescence resonance energy transfer measurements have been used to probe the structural and kinetic parameters of transfer RNA (tRNA) movements within the aminoacyl (A) and peptidyl (P) sites of the ribosome. Our investigation of tRNA motions, quantified on wild-type, mutant, and L1-depleted ribosome complexes, reveals a dynamic exchange between three metastable tRNA configurations, one of which is a previously unidentified hybrid state in which only deacylated-tRNA adopts its hybrid (P/E) configuration. This new dynamic information suggests a framework in which the formation of intermediate states in the translocation process is achieved through global conformational rearrangements of the ribosome particle.

Global conformational rearrangements during the activation of the GDP/GTP exchange factor Vav3

Activation of Rho/Rac GTPases during cell signaling requires the participation of GDP/GTP exchange factors of the Dbl family. Although the structure of the catalytic core of Dbl proteins has been established recently, the molecular changes that the full-length proteins experience during normal or oncogenic conditions of stimulation are still unknown. Here, we have used single-particle electron microscopy to solve the structures of the inactive (unphosphorylated), active (phosphorylated), and constitutively active (N-terminally deleted) versions of the exchange factor Vav3. Comparison of these forms has revealed the interdomain interactions maintaining the inactive Vav3 state and the dynamic changes that the overall Vav3 structure undergoes upon tyrosine phosphorylation. We have also found that the conformations of phosphorylated Vav3 and N-terminally deleted Vav3 are distinct, indicating that the acquisition of constitutive activity by exchange factors is structurally more complex than the mere elimination of inhibitory interactions between structural domains.

Near native structure in an RNA collapsed state.

Many large RNAs form conformationally collapsed, but non-native, states prior to folding to the native state or assembling with protein cofactors. Although RNA collapsed states play fundamental roles in RNA folding and ribonucleoprotein assembly processes, their structures have been poorly understood. We obtained 12 high-quality structural constraints for the collapsed state formed by the catalytic core of the bI5 intron RNA using site-specific cross-linking mediated by a short-lived reactant. RNA tertiary structures in the collapsed and native states are indistinguishable, even though only the native state forms a solvent-inaccessible core. Thus, structural neighbors in the collapsed state, including several long-range tertiary interactions, are approximately as close in space as in the native state, but RNA packing is sufficiently loose or dynamic to allow access by solvent. Binding by the obligate CBP2 protein cofactor has almost no effect on structural neighbors reported by cross-linking, even though protein binding chases the RNA from the collapsed state to the native state. Protein binding thus appears to promote only the final few angstroms of RNA folding rather than mediate global conformational rearrangements in the catalytic core. The bI5 RNA collapsed state functions to self-chaperone ribonucleoprotein assembly because this conformationally restrained structure lies very near that of the native state and excludes structures that otherwise misassemble efficiently.

Integrin Signals from Both Sides Now

Two new studies shed light on the mechanisms by which integrins "integrate" information from both sides of the plasma membrane. The integrins, a family of heterodimeric transmembrane receptors with large extracellular domains and small cytoplasmic tails, play a key role in transducing information about the extracellular matrix into responses affecting cell motility, growth, and survival (outside-in signaling). Cytoplasmic signals regulate integrin clustering and ligand-binding affinity to integrate these responses with information from other signaling pathways (inside-out signaling). Vinogradova et al. used nuclear magnetic resonance (NMR) to demonstrate interactions between the cytoplasmic tails of the integrin αIIb and β3 subunits and, using highly soluble fusion constructs, to determine the structure of the αIIb-β3 cytoplasmic tail complex. Interactions between the tail domains were abolished by mutations that constitutively activated the αIIbβ3 receptor and were disrupted by a cytoskeletal protein domain that enhanced ligand binding. Takagi et al. used electron microscopy, as well as analytical gel filtration, to demonstrate global conformational rearrangements of integrin αVβ3 extracellular domains under various activating and inactivating conditions. Solution-phase binding assays, combined with the mutational introduction of disulfide bonds into integrins expressed in cells, showed that conditions favoring a highly bent conformation were associated with a low affinity for ligands, whereas ligand binding or dissociation of the COOH termini favored an extended conformation with high affinity for ligands. These studies support a "switchblade" model of integrin activation, in which inside-out signals promote dissociation of the cytoplasmic tails, enhancing ligand affinity and leading to an intermediate conformation, which, like ligand binding itself, favors assumption of the high-affinity active form. O. Vinogradova, A. Velyvis, A. Velyviene, B. Hu, T. A. Haas, E. F. Plow, J. Qin, A structural mechanism of integrin α IIb β 3 "inside-out" activation as regulated by its cytoplasmic face. Cell 110 , 587-597 (2002). [Online Journal] J. Takagi, B. M. Petre, T. Watz, T. A. Springer, Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling. Cell 110 , 599-611 (2002). [Online Journal]

Integrin Signals from Both Sides Now

Two new studies shed light on the mechanisms by which integrins "integrate" information from both sides of the plasma membrane. The integrins, a family of heterodimeric transmembrane receptors with large extracellular domains and small cytoplasmic tails, play a key role in transducing information about the extracellular matrix into responses affecting cell motility, growth, and survival (outside-in signaling). Cytoplasmic signals regulate integrin clustering and ligand-binding affinity to integrate these responses with information from other signaling pathways (inside-out signaling). Vinogradova et al. used nuclear magnetic resonance (NMR) to demonstrate interactions between the cytoplasmic tails of the integrin αIIb and β3 subunits and, using highly soluble fusion constructs, to determine the structure of the αIIb-β3 cytoplasmic tail complex. Interactions between the tail domains were abolished by mutations that constitutively activated the αIIbβ3 receptor and were disrupted by a cytoskeletal protein domain that enhanced ligand binding. Takagi et al. used electron microscopy, as well as analytical gel filtration, to demonstrate global conformational rearrangements of integrin αVβ3 extracellular domains under various activating and inactivating conditions. Solution-phase binding assays, combined with the mutational introduction of disulfide bonds into integrins expressed in cells, showed that conditions favoring a highly bent conformation were associated with a low affinity for ligands, whereas ligand binding or dissociation of the COOH termini favored an extended conformation with high affinity for ligands. These studies support a "switchblade" model of integrin activation, in which inside-out signals promote dissociation of the cytoplasmic tails, enhancing ligand affinity and leading to an intermediate conformation, which, like ligand binding itself, favors assumption of the high-affinity active form.O. Vinogradova, A. Velyvis, A. Velyviene, B. Hu, T. A. Haas, E. F. Plow, J. Qin, A structural mechanism of integrin αIIbβ3 "inside-out" activation as regulated by its cytoplasmic face. Cell 110, 587-597 (2002). [Online Journal]J. Takagi, B. M. Petre, T. Watz, T. A. Springer, Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling. Cell 110, 599-611 (2002). [Online Journal]

Global Conformational Rearrangements in Integrin Extracellular Domains in Outside-In and Inside-Out Signaling

How ligand binding alters integrin conformation in outside-in signaling, and how inside-out signals alter integrin affinity for ligand, have been mysterious. We address this with electron microscopy, physicochemical measurements, mutational introduction of disulfides, and ligand binding to αVβ3 and αIIbβ3 integrins. We show that a highly bent integrin conformation is physiological and has low affinity for biological ligands. Addition of a high affinity ligand mimetic peptide or Mn 2+ results in a switchblade-like opening to an extended structure. An outward swing of the hybrid domain at its junction with the I-like domain shows conformational change within the headpiece that is linked to ligand binding. Breakage of a C-terminal clasp between the α and β subunits enhances Mn 2+-induced unbending and ligand binding.

The Alzheimer's Peptide Aβ Adopts a Collapsed Coil Structure in Water

Theself-assembly of the soluble peptide Aβ into Alzheimer's disease amyloid is believed to involve a conformational change. Hence the solution conformation of Aβ is of significant interest. In contrast to studies in other solvents, in water Aβ is collapsed into a compact series of loops, strands, and turns and has no α-helical or β-sheet structure. Conformational stabilization is primarily attributed to van der Waals and electrostatic forces. A large conspicuous uninterrupted hydrophobic patch covers ∼25% of the surface. The compact coil structure appears meta-stable, and because fibrillization leads to formation of intermolecular β-sheet secondary structure, a global conformational rearrangement is highly likely. A molecular hypothesis for amyloidosis includes at least two primary driving forces, changes in solvation thermodynamics during formation of amyloid deposits and relief of internal conformational stress within the soluble precursor during formation of lower-energy amyloid fibrils.

Hydration of Biological Macromolecules in Solution: Surface Structure and Molecular Recognition

The presently available three-dimensional protein and nucleic acid structures at atomic resolution have, with few exceptions, been determined either by X-ray diffraction in protein crystals (Blundell and Johnson 1976) or by nuclear magnetic resonance (NMR) spectroscopy in protein solutions (Wuthrich 1986). These two techniques produce a rapidly growing pool of data (Hendrickson and Wuthrich 1991, 1992, 1993), which represent today's structural basis for detailed investigations on the functionality of biological macromolecules. NMR spectroscopy and X-ray diffraction can provide complementary information on the same molecule, which results primarily from the facts that the time scales of the two types of measurements are widely different, and that the two techniques use, respectively, proteins in solution and protein single crystals (see, e.g., Wuthrich 1990, 1991). Since protein structure determinations by NMR or by X-ray diffraction can be performed independently (Blundell and Johnson 1976; Wuthrich 1986), meaningful comparisons of corresponding structures in single crystals and in noncrystalline states can be obtained. This is highly relevant, since the solution conditions for NMR studies may coincide closely with the physiological fluids, and comparative studies of corresponding crystal and solution structures promise to result in more relevant ways of analyzing crystal data with regard to protein functions in physiological milieus. Providing this kind of fundamental information may well turn out to be the major impact of NMR in structural biology, considering that X-ray crystallography continues to provide a dominant fraction of the new macromolecular structures (Hendrickson and Wuthrich 1993). Although NMR structure determination in solution will foreseeably be limited to proteins with molecular weights below about 30,000-40,000 (see, e.g., Wuthrich 1990), many conclusions from comparative studies with relatively small proteins should also be applicable to crystal structures of bigger molecules. In comparisons of corresponding crystal and solution structures of proteins, both global conformational rearrangements and extensive conservation between the two states have been observed. Major rearrangements are usually seen in nonglobular polypeptides (see, e.g., Braun et al. 1983) and on the surface of globular proteins, whereas close coincidence is commonly encountered for the core of globular proteins (Billeter 1992). However, even for proteins with virtually identical molecular architecture in crystals and in solution, the two techniques provide different information on the internal mobility of the molecular core. NMR can provide direct, quantitative measurements of the frequencies of certain high-activation-energy motional processes in the interior of globular proteins, and at least semiquantitative information on additional, higher-frequency processes. The corresponding information from X-ray structure determinations commonly consists of an outline of the conformation space covered by the combination of static disorder and high-frequency structure fluctuations. The protein surface is most likely to be influenced by crystal packing, or by solvent interactions, respectively, and it is thus of special interest to investigate these effects by combined use of NMR and crystallography. On the one hand, a detailed description of the protein surface in near-physiological solution represents a proper reference for investigating mechanistic aspects of intermolecular interactions with proteins, and novel NMR techniques enabling the observation of hydration water on the molecular surface (Otting et al. 1991a) have greatly added to the characterization of protein surfaces in solution. On the other hand, the structural basis of intermolecular recognition between different macromolecules can be investigated in a large sample of X-ray crystal structures of proteins in binary complexes or multimolecular assemblies, and even the influence of crystal-packing effects on the protein surface may be indicative of the types of protein-protein interactions that are important in physiological recognition processes. In this paper, I survey novel insights into protein structures in solution that result from NMR investigations of protein hydration. Special emphasis is on protein surface hydration in aqueous solution and comparison with corresponding data from diffraction experiments with protein crystals. As an illustration, hydration data on a homeodomain-DNA complex (Billeter et al. 1993; Qian et al. 1993a) are discussed.

Protein conformational perturbations affect the photoreduction of native cytochrome c peroxidase (III) at alkaline pH.

Ferric cytochrome c peroxidase (CCP) undergoes a ligation-state transition from a pentacoordinate, high-spin (5c/hs) heme to a hexacoordinate, low-spin (6c/1s) heme when titrated over a pH range of 7.30-9.70. This behavior is similar to that exhibited by the ferrous form of the enzyme. However, the photodissociation of the low-spin, axial ligand, exhibited by ferrous CCP at alkaline pH, is not observed for ferric CCP. Instead, a photoinduced reduction of the ferric heme is apparent in the pH range 7.90-9.70. In the absence of O2 and redox mediators such as methyl viologen (MV2+), the reoxidation of the photoreduced enzyme is very slow (tau 1/2 approximately 3 min). F(-)-bound CCP(III) (6c/hs) displays similar pH-dependent photoreduction. Horseradish peroxidase, however, does not. The formation of 6c/1s heme coincides with the onset of appreciable photoreduction (between laser pulses, > 60 ms) of CCP (III) at alkaline pH, suggesting a global protein conformational rearrangement within or around its heme pocket. Photoreduction of alkaline CCP(III) most likely involves intramolecular electron transfer (ET) from the aromatic residue in the proximal heme pocket to the photoexcited heme. We speculate that the kinetics of electron transfer are affected by changes in the orientation of Trp-191.