The Folding Trajectory of RNase H Is Dominated by Its Topology and Not Local Stability: A Protein Engineering Study of Variants that Fold via Two-State and Three-State Mechanisms

Proteins can sample a variety of partially folded conformations during the transition between the unfolded and native states. Some proteins never significantly populate these high-energy states and fold by an apparently two-state process. However, many proteins populate detectable, partially folded forms during the folding process. The role of such intermediates is a matter of considerable debate. A single amino acid change can convert Escherichia coli ribonuclease H from a three-state folder that populates a kinetic intermediate to one that folds in an apparent two-state fashion. We have compared the folding trajectories of the three-state RNase H and the two-state RNase H, proteins with the same native-state topology but altered regional stability, using a protein engineering approach. Our data suggest that both versions of RNase H fold through a similar trajectory with similar high-energy conformations. Mutations in the core and the periphery of the protein affect similar aspects of folding for both variants, suggesting a common trajectory with folding of the core region followed by the folding of the periphery. Our results suggest that formation of specific partially folded conformations may be a general feature of protein folding that can promote, rather than hinder, efficient folding.     

Dynamic and Thermodynamic Response of the Ras Protein Cdc42Hs upon Association with the Effector Domain of PAK3

Human cell division cycle protein 42 (Cdc42Hs) is a small, Rho-type guanosine triphosphatase involved in multiple cellular processes through its interactions with downstream effectors. The binding domain of one such effector, the actin cytoskeleton-regulating p21-activated kinase 3, is known as PBD46. Nitrogen-15 backbone and carbon-13 methyl NMR relaxation was measured to investigate the dynamical changes in activated GMPPCP·Cdc42Hs upon PBD46 binding. Changes in internal motion of the Cdc42Hs, as revealed by methyl axis order parameters, were observed not only near the Cdc42Hs–PBD46 interface but also in remote sites on the Cdc42Hs molecule. The binding-induced changes in side-chain dynamics propagate along the long axis of Cdc42Hs away from the site of PBD46 binding with sharp distance dependence. Overall, the binding of the PBD46 effector domain on the dynamics of methyl-bearing side chains of Cdc42Hs results in a modest rigidification, which is estimated to correspond to an unfavorable change in conformational entropy of approximately − 10 kcal mol^− 1 at 298 K. A cluster of methyl probes closest to the nucleotide-binding pocket of Cdc42Hs becomes more rigid upon binding of PBD46 and is proposed to slow the catalytic hydrolysis of the γ phosphate moiety. An additional cluster of methyl probes surrounding the guanine ring becomes more flexible on binding of PBD46, presumably facilitating nucleotide exchange mediated by a guanosine exchange factor. In addition, the Rho insert helix, which is located at a site remote from the PBD46 binding interface, shows a significant dynamic response to PBD46 binding.     

Subsequent events to GTP binding by the plant PsbO protein: Structural changes, GTP hydrolysis and dissociation from the photosystem II complex

Besides an essential role in optimizing water oxidation in photosystem II (PSII), it has been reported that the spinach PsbO protein binds GTP [C. Spetea, T. Hundal, B. Lundin, M. Heddad, I. Adamska, B. Andersson, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 1409-1414]. Here we predict four GTP-binding domains in the structure of spinach PsbO, all localized in the β-barrel domain of the protein, as judged from comparison with the 3D-structure of the cyanobacterial counterpart. These domains are not conserved in the sequences of the cyanobacterial or green algae PsbO proteins. MgGTP induces specific changes in the structure of the PsbO protein in solution, as detected by circular dichroism and intrinsic fluorescence spectroscopy. Spinach PsbO has a low intrinsic GTPase activity, which is enhanced fifteen-fold when the protein is associated with the PSII complex in its dimeric form. GTP stimulates the dissociation of PsbO from PSII under light conditions known to also release Mn²⁺ and Ca²⁺ ions from the oxygen-evolving complex and to induce degradation of the PSII reaction centre D1 protein. We propose the occurrence in higher plants of a PsbO-mediated GTPase activity associated with PSII, which has consequences for the function of the oxygen-evolving complex and D1 protein turnover.     

Role of the alpha(1) integrin cytoplasmic tail in the formation of focal complexes, actin organization, and in the control of cell migration

Integrins play a key role in cellular motility; an essential process for embryonic development and tissue morphogenesis, and also for pathological processes such as tumor cell invasion and metastasis. Recently, we showed that the cytoplasmic tail of integrin alpha(1) regulates the formation of focal complexes, F-actin cytoskeleton reorganization, and migration. We now report that the alpha(1) tail directly engages in collagen IV-mediated migration by regulation of the small GTPase Rac1. Deletion variants of the alpha(1) integrin differ in their ability to activate Rac1. Constitutively active Rac1 rescues motility in otherwise immotile cells expressing a truncated alpha(1) integrin without any cytoplasmic tail. In these cells, levels of GTP-Rac1 are constitutively elevated, but kept non-functional in the cytoplasm. The conserved GFFKR motif is sufficient to convey Rac1 activation, but downregulates the amount of GTP-Rac1 in the absence of the alpha(1)-specific sequence PLKKKMEK. This sequence is also required for the recruitment of PI3K to focal adhesions following Rac1 activation. Our results demonstrate that the short alpha(1) cytoplasmic tail is crucial for Rac1 activation and PI3K localization, which in turn results in cytoskeletal rearrangement and subsequent migration.     

Structural Analysis of the Interactions Between Hsp70 Chaperones and the Yeast DNA Replication Protein Orc4p

Hsp70 chaperones, besides their role in assisting protein folding, are key modulators of protein disaggregation, being consistently found as components of most macromolecular assemblies isolated in proteome-wide affinity purifications. A wealth of structural information has been recently acquired on Hsp70s complexed with Hsp40 and NEF co-factors and with small hydrophobic target peptides. However, knowledge of how Hsp70s recognize large protein substrates is still limited. Earlier, we reported that homologue Hsp70 chaperones (DnaK in Escherichia coli and Ssa1-4p/Ssb1-2p in Saccharomyces cerevisiae) bind strongly, both in vitro and in vivo, to the AAA+ domain in the Orc4p subunit of yeast origin recognition complex (ORC). ScORC is the paradigm for eukaryotic DNA replication initiators and consists of six distinct protein subunits (ScOrc1p-ScOrc 6p). Here, we report that a hydrophobic sequence (IL₄) in the initiator specific motif (ISM) in Orc4p is the main target for DnaK/Hsp70. The three-dimensional electron microscopy reconstruction of a stable Orc4p₂-DnaK complex suggests that the C-terminal substrate-binding domain in the chaperone clamps the AAA+ IL₄ motif in one Orc4p molecule, with the substrate-binding domain lid subdomain wedging apart the other Orc4p subunit. Pairwise co-expression in E. coli shows that Orc4p interacts with Orc1/2/5p. Mutation of IL₄ selectively disrupts Orc4p interaction with Orc2p. Allelic substitution of ORC4 by mutants in each residue of IL₄ results in lethal (I184A) or thermosensitive (L185A and L186A) initiation-defective phenotypes in vivo. The interplay between Hsp70 chaperones and the Orc4p-IL₄ motif might have an adaptor role in the sequential, stoichiometric assembly of ScORC subunits.     

Compensatory and long-range changes in picosecond-nanosecond main-chain dynamics upon complex formation: 15N relaxation analysis of the free and bound states of the ubiquitin-like domain of human plexin-B1 and the small GTPase Rac1

The formation of a complex between Rac1 and the cytoplasmic domain of plexin-B1 is one of the first documented cases of a direct interaction between a small guanosine 5′-triphosphatase (GTPase) and a transmembrane receptor. Structural studies have begun to elucidate the role of this interaction for the signal transduction mechanism of plexins. Mapping of the Rac1 GTPase surface that contacts the Rho GTPase binding domain of plexin-B1 by solution NMR spectroscopy confirms the plexin domain as a GTPase effector protein. Regions neighboring the GTPase switch I and II regions are also involved in the interaction and there is considerable interest to examine the changes in protein dynamics that take place upon complex formation. Here we present main-chain nitrogen-15 relaxation measurements for the unbound proteins as well as for the Rho GTPase binding domain and Rac1 proteins in their complexed state. Derived order parameters, S², show that considerable motions are maintained in the bound state of plexin. In fact, some of the changes in S² on binding appear compensatory, exhibiting decreased as well as increased dynamics. Fluctuations in Rac1, already a largely rigid protein on the picosecond-nanosecond timescale, are overall diminished, but isomerization dynamics in the switch I and II regions of the GTPase are retained in the complex and appear to be propagated to the bound plexin domain. Remarkably, fluctuations in the GTPase are attenuated at sites, including helices α6 (the Rho-specific insert helix), α7 and α8, that are spatially distant from the interaction region with plexin. This effect of binding on long-range dynamics appears to be communicated by hinge sites and by subtle conformational changes in the protein. Similar to recent studies on other systems, we suggest that dynamical protein features are affected by allosteric mechanisms. Altered protein fluctuations are likely to prime the Rho GTPase-plexin complex for interactions with additional binding partners.     

Molecular Determinants Required for Selective Interactions between the Thyroid Hormone Receptor Homodimer and the Nuclear Receptor Corepressor N-CoR

The unliganded nuclear receptor (NR) generally recruits the NR corepressor (N-CoR) and the silencing mediator of retinoid and thyroid hormone receptor via its direct binding to the extended helical motif within dual NR-interaction domains (IDs) of corepressors. Interestingly, N-CoR has a third ID (ID3) upstream of two IDs (ID1 and ID2) and its core motif (IDVII), rather than an extended helical motif, is known to be involved directly in the exclusive interaction of ID3 with the thyroid hormone receptor (TR). Here, we investigated the molecular determinants of the TR interaction with ID3 to understand the molecular basis of the N-CoR preference shown by the TR homodimer. Using a one- plus two-hybrid system, we identified the specific residues of N-CoR-ID2 and N-CoR-ID3 that are required for stable association of N-CoR with the TR homodimer. By swapping experiments and mutagenesis studies, we found that the C-terminally flanked residues of the core motif of ID3 contribute to the TR preference for N-CoR-ID3, suggesting that an extended three-turn helix might form within the ID3 via a C-terminal extension (IDVIITRQI) and participate directly in the TR-specific interaction. Structural modeling of the ID3 motif on TR-LBD is consistent with this conclusion. Notably, we identified a novel interaction between N-CoR-ID3 and orphan NR RevErb that is mediated by the residues crucial also in TR binding. These observations raise the intriguing possibility that NR homodimers such as TR and RevErb display preferential binding to the N-CoR corepressor via their specific interactions with ID3, which is normally absent from the silencing mediator of retinoid and thyroid hormone receptor.     

Structural Evidence for a Sequential Release Mechanism for Activation of Heterotrimeric G Proteins

Heptahelical G-protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors couple to heterotrimeric G proteins to relay extracellular signals to intracellular signaling networks, but the molecular mechanism underlying guanosine 5′-diphosphate (GDP) release by the G protein α-subunit is not well understood. Amino acid substitutions in the conserved α5 helix of G_i, which extends from the C-terminal region to the nucleotide-binding pocket, cause dramatic increases in basal (receptor-independent) GDP release rates. For example, mutant Gα_i1-T329A shows an 18-fold increase in basal GDP release rate and, when expressed in culture, it causes a significant decrease in forskolin-stimulated cAMP accumulation. The crystal structure of Gα_i1-T329A·GDP shows substantial conformational rearrangement of the switch I region and additional striking alterations of side chains lining the catalytic pocket that disrupt the Mg⁺² coordination sphere and dislodge bound Mg⁺². We propose a “sequential release” mechanism whereby a transient conformational change in the α5 helix alters switch I to induce GDP release. Interestingly, this mechanistic model for heterotrimeric G protein activation is similar to that suggested for the activation of the plant small G protein Rop4 by RopGEF8.     

Interaction of activated Rab5 with actin-bundling proteins, L- and T-plastin and its relevance to endocytic functions in mammalian cells

Rab5 is a GTP-binding protein that is crucial for endocytic machinery functions. We previously identified L-plastin as a binding protein for Rab5, using an affinity column with constitutively active Rab5. L- and T-plastin are isoforms of a plastin protein family belonging to actin-bundling proteins that are implicated in the regulation of cell morphology, lamellipodium protrusion, bacterial invasion and tumor progression. However, the physiological relevance of Rab5 binding to plastin has remained unclear. Here, we show that L- and T-plastin interacted only with activated Rab5 and that they co-localized with Rab5 on the plasma membrane and endosome. Rab5 activity was also higher in both L- and T-plastin over-expressing Cos-1 cells. Furthermore, expression of L- and T-plastin increased the rate of fluid-phase endocytosis. These findings imply that the Rab5 is either activated or the activity is sustained by interaction with plastin, and that this interaction influences endocytic activity.     

Molecular basis of guanine nucleotide dissociation inhibitor activity of human neuroglobin by chemical cross-linking and mass spectrometry

Oxidized human neuroglobin (Ngb), a heme protein expressed in the brain, has been proposed to act as a guanine nucleotide dissociation inhibitor (GDI) for the GDP-bound form of the heterotrimeric G protein alpha-subunit (Galpha(i)). Here, to elucidate the molecular mechanism underlying the GDI activity of Ngb, we used an glutathione-S-transferase pull-down assay to confirm that Ngb competes with G-protein betagamma-subunits (Gbetagamma) for binding to Galpha(i), and identified the Galpha(i)-binding site in Ngb by chemical cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and sulfo-N-hydroxysuccinimide, coupled with mass spectrometry (MS). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis for tryptic peptides derived from the cross-linked Ngb-Galpha(i) complex revealed several binding regions in Ngb. Furthermore, MALDI-TOF/TOF MS analysis of the cross-linked Ngb and Galpha(i) peptides, together with the MS/MS scoring method, predicted cross-linking between Glu60 (Ngb) and Ser206 (Galpha(i)), and between Glu53 (Ngb) and Ser44 (Galpha(i)). Because Ser206 of Galpha(i) is located in the region that contacts Gbetagamma, binding of Ngb could facilitate the release of Gbetagamma from Galpha(i). Binding of Ngb to Galpha(i) would also inhibit the exchange of GDP for GTP, because Ser44 (Galpha(i)) is adjacent to the GDP-binding site and Glu53 (Ngb), which is cross-linked to Ser44 (Galpha(i)), could be located close to GDP. Thus, we have identified, for the first time, the sites of interaction between Ngb and Galpha(i), enabling us to discuss the functional significance of this binding on the GDI activity of Ngb.     

GEFs, GAPs, GDIs and effectors: taking a closer (3D) look at the regulation of Ras-related GTP-binding proteins

Cell biology depends on the interactions of macromolecules, such as protein—DNA, protein—protein or protein—nucleotide interactions. GTP-binding proteins are no exception to the rule. They regulate cellular processes as diverse as protein biosynthesis and intracellular membrane trafficking. Recently, a large number of genes encoding GTP-binding proteins and the proteins that interact witht these molecular switches have been cloned and expressed. The 3D structures of some of these have also been elucidated     

Biochemical and Structural Study of the Homologues of the Thiol-Disulfide Oxidoreductase DsbA in Neisseria meningitidis

Bacterial virulence depends on the correct folding of surface-exposed proteins, a process catalyzed by the thiol-disulfide oxidoreductase DsbA, which facilitates the synthesis of disulfide bonds in Gram-negative bacteria. The Neisseria meningitidis genome possesses three genes encoding active DsbAs: DsbA1, DsbA2 and DsbA3. DsbA1 and DsbA2 have been characterized as lipoproteins involved in natural competence and in host interactive biology, while the function of DsbA3 remains unknown.This work reports the biochemical characterization of the three neisserial enzymes and the crystal structures of DsbA1 and DsbA3. As predicted by sequence homology, both enzymes adopt the classic Escherichia coli DsbA fold. The most striking feature shared by all three proteins is their exceptional oxidizing power. With a redox potential of − 80 mV, the neisserial DsbAs are the most oxidizing thioredoxin-like enzymes known to date. Consistent with these findings, thermal studies indicate that their reduced form is also extremely stable. For each of these enzymes, this study shows that a threonine residue found within the active-site region plays a key role in dictating this extraordinary oxidizing power. This result highlights how residues located outside the CXXC motif may influence the redox potential of members of the thioredoxin family.     

Functional characterization of fish neuroglobin: Zebrafish neuroglobin is highly expressed in amacrine cells after optic nerve injury and can translocate into ZF4 cells

Neuroglobin (Ngb) is a recently discovered vertebrate heme protein that is expressed in the brain and can reversibly bind oxygen. Mammalian Ngb is involved in neuroprotection under conditions of oxidative stress, such as ischemia and reperfusion. We previously found that zebrafish Ngb can penetrate the mammalian cell membrane. In the present study, we investigated the functional characteristics of fish Ngb by using the zebrafish cell line ZF4 and zebrafish retina. We found that zebrafish Ngb translocates into ZF4 cells, but cannot protect ZF4 cells against cell death induced by hydrogen peroxide. Furthermore, we demonstrated that a chimeric ZHHH Ngb protein, in which module M1 of human Ngb is replaced by that of zebrafish, is a cell-membrane-penetrating protein that can protect ZF4 cells against hydrogen peroxide exposure. Moreover, we investigated the localization of Ngb mRNA and protein in zebrafish retina and found that Ngb mRNA is expressed in amacrine cells in the inner nuclear layer and is significantly increased in amacrine cells 3 days after optic nerve injury. Immunohistochemical studies clarified that Ngb protein levels were increased in both amacrine cells and presynaptic regions in the inner plexiform layer after nerve injury. Taken together, we hypothesize that fish Ngb, whose expression is upregulated in amacrine cells after optic nerve injury, might be released from amacrine cells, translocate into neighboring ganglion cells, and function in the early stage of optic nerve regeneration. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Hypoglycosylation of dystroglycan due to T192M mutation: A molecular insight behind the fact

Abnormal glycosylation of dystroglycan (DG), a transmembrane glycoprotein, results in a group of diseases known as dystroglycanopathy. A severe dystroglycanopathy known as the limb girdle disease MDDGC9 [OMIM: 613818] occurs as a result of hypoglycosylation of alpha subunit of DG. Reasons behind this has been traced back to a point mutation (T192M) in DG that leads to weakening of interactions of DG protein with laminin and subsequent loss of signal flow through the DG protein. In this work we have tried to analyze the molecular details of the interactions between DG and laminin1 in order to propose a mechanism about the onset of the disease MDDGC9. We have observed noticeable changes between the modeled structures of wild type and mutant DG proteins. We also have employed molecular docking techniques to study and compare the binding interactions between laminin1 and both the wild type and mutant DG proteins. The docking simulations have revealed that the mutant DG has weaker interactions with laminin1 as compared to the wild type DG. Till date there are no previous reports that deal with the elucidation of the interactions of DG with laminin1 from the molecular level. Our study is therefore the first of its kind which analyzes the differences in binding patterns of laminin1 with both the wild type and mutant DG proteins. Our work would therefore facilitate analysis of the molecular mechanism of the disease MDDGC9. Future work based on our results may be useful for the development of suitable drugs against this disease.     

Disease-associated Substitutions in the Filamin B Actin Binding Domain Confer Enhanced Actin Binding Affinity in the Absence of Major Structural Disturbance: Insights from the Crystal Structures of Filamin B Actin Binding Domains

Missense mutations in filamin B (FLNB) are associated with the autosomal dominant atelosteogenesis (AO) and the Larsen group of skeletal malformation disorders. These mutations cluster in particular FLNB protein domains and act in a presumptive gain-of-function mechanism. In contrast the loss-of-function disorder, spondylocarpotarsal synostosis syndrome, is characterised by the complete absence of FLNB. One cluster of AO missense mutations is found within the second of two calponin homology (CH) domains that create a functional actin-binding domain (ABD). This N-terminal ABD is required for filamin F-actin crosslinking activity, a crucial aspect of filamin's role of integrating cell-signalling events with cellular scaffolding and mechanoprotection. This study characterises the wild type FLNB ABD and investigates the effects of two disease-associated mutations on the structure and function of the FLNB ABD that could explain a gain-of-function mechanism for the AO diseases. We have determined high-resolution X-ray crystal structures of the human filamin B wild type ABD, plus W148R and M202V mutants. All three structures display the classic compact monomeric conformation for the ABD with the CH1 and CH2 domains in close contact. The conservation of tertiary structure in the presence of these mutations shows that the compact ABD conformation is stable to the sequence substitutions. In solution the mutant ABDs display reduced melting temperatures (by 6-7 °C) as determined by differential scanning fluorimetry. Characterisation of the wild type and mutant ABD F-actin binding activities via co-sedimentation assays shows that the mutant FLNB ABDs have increased F-actin binding affinities, with dissociation constants of 2.0 μM (W148R) and 0.56 μM (M202V), compared to the wild type ABD K_d of 7.0 μM. The increased F-actin binding affinity of the mutants presents a biochemical mechanism that differentiates the autosomal dominant gain-of-function FLNB disorders from those that arise through the complete loss of FLNB protein.     

Structure of the Complex between HER2 and an Antibody Paratope Formed by Side Chains from Tryptophan and Serine

Engineered antibody paratopes with limited sequence diversity permit assessment of the roles played by different amino acid side chains in creating the high-affinity, high-specificity interactions characteristic of antibodies. We describe a paratope raised against the human ErbB family member HER2, using a binary diversity tryptophan/serine library displayed on phage. Fab37 binds to the extracellular domain of HER2 with sub-nanomolar affinity. An X-ray structure at 3.2 Å resolution reveals a contact paratope composed almost entirely of tryptophan and serine residues. Mutagenesis experiments reveal which of these side chains are more important for direct antigen interactions and which are more important for conformational flexibility. The crystal lattice contains an unprecedented trimeric arrangement of HER2 closely related to previously observed homodimers of the related epidermal growth factor receptor.     

G proteins, effectors and GAPs: structure and mechanism

G proteins form a diverse family of regulatory GTPases which, in the GTP-bound state, bind to and activate downstream effectors. Structure of Ras homologs bound to effector domains have revealed mechanisms by which G proteins couple GTP binding to effector activation and achieve specificity. Complexes between structurally unrelated GTPase-activating proteins with complementary G proteins suggest common mechanisms by which GTP hydrolysis is stimulated via direct interactions with conformationally labile switch regions of the G protein.