Improved structural characterizations of the drkN SH3 domain unfolded state suggest a compact ensemble with native-like and non-native structure

Due to their dynamic ensemble nature and a deficiency of experimental restraints, disordered states of proteins are difficult to characterize structurally. Here, we have expanded upon our previous work on the unfolded state of the Drosophila drk N-terminal (drkN) SH3 domain with our program ENSEMBLE, which assigns population weights to pregenerated conformers in order to calculate ensembles of structures whose properties are collectively consistent with experimental measurements. The experimental restraint set has been enlarged with newly measured paramagnetic relaxation enhancements from Cu(2+) bound to an amino terminal Cu(2+)-Ni(2+) binding (ATCUN) motif as well as nuclear Overhauser effect (NOE) and hydrogen exchange data from recent studies. In addition, two new pseudo-energy minimization algorithms have been implemented that have dramatically improved the speed of ENSEMBLE population weight assignment. Finally, we have greatly improved our conformational sampling by utilizing a variety of techniques to generate both random structures and structures that are biased to contain elements of native-like or non-native structure. Although it is not possible to uniquely define a representative structural ensemble, we have been able to assess various properties of the drkN SH3 domain unfolded state by performing ENSEMBLE minimizations of different conformer pools. Specifically, we have found that the experimental restraint set enforces a compact structural distribution that is not consistent with an overall native-like topology but shows preference for local non-native structure in the regions corresponding to the diverging turn and the beta5 strand of the folded state and for local native-like structure in the region corresponding to the beta6 and beta7 strands. We suggest that this approach could be generally useful for the structural characterization of disordered states.     

Structure, Dynamics and Folding of an Immunoglobulin Domain of the Gelation Factor (ABP-120) from Dictyostelium discoideum

We have carried out a detailed structural and dynamical characterisation of the isolated fifth repeat of the gelation factor (ABP-120) from Dictyostelium discoideum (ddFLN5) by NMR spectroscopy to provide a basis for studies of co-translational folding on the ribosome of this immunoglobulin-like domain. The isolated ddFLN5 can fold autonomously in solution into a structure that resembles very closely the crystal structure of the domain in a construct in which the adjacent sixth repeat (ddFLN6) is covalently linked to its C-terminus in tandem but deviates locally from a second crystal structure in which ddFLN5 is flanked by ddFLN4 and ddFLN6 at both N- and C-termini. Conformational fluctuations were observed via ¹⁵N relaxation methods and are primarily localised in the interstrand loops that encompass the C-terminal hemisphere. These fluctuations are distinct in location from the region where line broadening is observed in ddFLN5 when attached to the ribosome as part of a nascent chain. This observation supports the conclusion that the broadening is associated with interactions with the ribosome surface [Hsu, S. T. D., Fucini, P., Cabrita, L. D., Launay, H., Dobson, C. M. & Christodoulou, J. (2007). Structure and dynamics of a ribosome-bound nascent chain by NMR spectroscopy. Proc. Natl. Acad. Sci. USA, 104, 16516-16521]. The unfolding of ddFLN5 induced by high concentrations of urea shows a low population of a folding intermediate, as inferred from an intensity-based analysis, a finding that differs from that of ddFLN5 as a ribosome-bound nascent chain. These results suggest that interesting differences in detail may exist between the structure of the domain in isolation and when linked to the ribosome and between protein folding in vitro and the folding of a nascent chain as it emerges from the ribosome.     

Backbone Dynamics and Global Effects of an Activating Mutation in Minimized Mtu RecA Inteins

Inteins mediate protein splicing, which has found many applications in biotechnology and protein engineering. A single valine-to-leucine mutation (V67L) can globally enhance splicing and related cleavage reactions in minimized Mycobacterium tuberculosis RecA inteins. However, V67L mutation causes little change in crystal structures. To test whether protein dynamics contribute to activity enhancement in the V67L mutation, we have studied the conformations and dynamics of the minimized and engineered intein ΔΔIhh-V67CM and a single V67L mutant, ΔΔIhh-L67CM, by solution NMR. Chemical shift perturbations established that the V67L mutation causes global changes, including changes at the N-terminus and C-terminus of the intein, which are active sites for protein splicing. The single V67L mutation significantly slows hydrogen-exchange rates globally, indicating a shift to more stable conformations and reduction in ensemble distribution. Whereas the V67L mutation causes little change for motions on the picosecond-to-nanosecond timescale, motions on the microsecond-to-millisecond timescale affect a region involving the conserved F-block histidine and C-terminal asparagine, which are residues important for C-terminal cleavage. The V67L mutation is proposed to activate splicing by reducing the ensemble distribution of the intein structure and by modifying the active sites.     

Molecular structure and metal-binding properties of the periplasmic CopK protein expressed in Cupriavidus metallidurans CH34 during copper challenge

The copK gene is localized on the pMOL30 plasmid of Cupriavidus metallidurans CH34 within the complex cop cluster of genes, for which 21 genes have been identified. The expression of the corresponding periplasmic CopK protein is strongly upregulated in the presence of copper, leading to a high periplasmic accumulation. The structure and metal-binding properties of CopK were investigated by NMR and mass spectrometry. The protein is dimeric in the apo state with a dissociation constant in the range of 10^- 5 M estimated from analytical ultracentrifugation. Mass spectrometry revealed that CopK has two high-affinity Cu(I)-binding sites per monomer with different Cu(I) affinities. Binding of Cu(II) was observed but appeared to be non-specific. The solution structure of apo-CopK revealed an all-β fold formed of two β-sheets in perpendicular orientation with an unstructured C-terminal tail. The dimer interface is formed by the surface of the C-terminal β-sheet. Binding of the first Cu(I)-ion induces a major structural modification involving dissociation of the dimeric apo-protein. Backbone chemical shifts determined for the 1Cu(I)-bound form confirm the conservation of the N-terminal β-sheet, while the last strand of the C-terminal sheet appears in slow conformational exchange. We hypothesize that the partial disruption of the C-terminal β-sheet is related to dimer dissociation. NH-exchange data acquired on the apo-protein are consistent with a lower thermodynamic stability of the C-terminal sheet. CopK contains seven methionine residues, five of which appear highly conserved. Chemical shift data suggest implication of two or three methionines (Met54, Met38, Met28) in the first Cu(I) site. Addition of a second Cu(I) ion further increases protein plasticity. Comparison of the structural and metal-binding properties of CopK with other periplasmic copper-binding proteins reveals two conserved features within these functionally related proteins: the all-β fold and the methionine-rich Cu(I)-binding site.     

Structural characterization of the intrinsically unfolded protein beta-synuclein, a natural negative regulator of alpha-synuclein aggregation

The synuclein family of intrinsically unfolded proteins is composed of three highly homologous members, alpha-synuclein (alphaS), beta-synuclein (betaS) and gamma-synuclein (gammaS), which are linked to neurodegenerative disorders and cancer. alphaS has been studied intensively after its identification as the major protein component of amyloid-like deposits in Parkinson's disease and dementia with Lewy bodies. betaS, on the other hand, was found to act as a potent inhibitor of alphaS amyloid formation, and it is proposed as a natural regulator of its neurotoxicity. It is then of particular interest to elucidate the structural and dynamic features of the soluble state of betaS as a first step to understand the molecular basis of its anti-amyloidogenic effect on alphaS. We present here the characterization of natively unstructured betaS by high resolution heteronuclear NMR techniques. A combination of pulse-field gradient, three-dimensional heteronuclear correlation, residual dipolar couplings, paramagnetic relaxation enhancement and backbone relaxation experiments were employed to characterize the ensemble of conformations populated by the protein. The results indicate that betaS adopts extended conformations in its native state, characterized by the lack of the long-range contacts as previously reported for alphaS. Despite the lack of defined secondary structure, we found evidence for transient polyproline II conformations clustered at the C-terminal region. The structuring of the backbone at the C terminus is locally encoded, stabilized by the presence of eight proline residues embedded in a polypeptide stretch rich in hydrophilic and negatively charged amino acids. The structural and functional implications of these findings are analyzed via a thorough comparison with its neurotoxic homolog alphaS.     

Structure of a Double Transmembrane Fragment of a G-Protein-Coupled Receptor in Micelles

The structure and dynamic properties of an 80-residue fragment of Ste2p, the G-protein-coupled receptor for α-factor of Saccharomyces cerevisiae, was studied in LPPG micelles with the use of solution NMR spectroscopy. The fragment Ste2p(G31-T110) (TM1-TM2) consisted of 19 residues from the N-terminal domain, the first TM helix (TM1), the first cytoplasmic loop, the second TM helix (TM2), and seven residues from the first extracellular loop. Multidimensional NMR experiments on [¹⁵N], [¹⁵N, ¹³C], [¹⁵N, ¹³C, ²H]-labeled TM1-TM2 and on protein fragments selectively labeled at specific amino acid residues or protonated at selected methyl groups resulted in >95% assignment of backbone and side-chain nuclei. The NMR investigation revealed the secondary structure of specific residues of TM1-TM2. TALOS constraints and NOE connectivities were used to calculate a structure for TM1-TM2 that was highlighted by the presence of three α-helices encompassing residues 39-47, 49-72, and 80-103, with higher flexibility around the internal Arg⁵⁸ site of TM1. RMSD values of individually superimposed helical segments 39-47, 49-72, and 80-103 were 0.25 ± 0.10 Å, 0.40 ± 0.13 Å, and 0.57 ± 0.19 Å, respectively. Several long-range interhelical connectivities supported the folding of TM1-TM2 into a tertiary structure typified by a crossed helix that splays apart toward the extracellular regions and contains considerable flexibility in the G⁵⁶VRSG⁶⁰ region. ¹⁵N-relaxation and hydrogen-deuterium exchange data support a stable fold for the TM parts of TM1-TM2, whereas the solvent-exposed segments are more flexible. The NMR structure is consistent with the results of biochemical experiments that identified the ligand-binding site within this region of the receptor.     

Solution structure and dynamics of the N-terminal cytosolic domain of rhomboid intramembrane protease from Pseudomonas aeruginosa: insights into a functional role in intramembrane proteolysis

Rhomboids are ubiquitous integral membrane proteases that release cellular signals from membrane-bound substrates through a general signal transduction mechanism known as regulated intramembrane proteolysis (RIP). We present the NMR structure of the cytosolic N-terminal domain (NRho) of P. aeruginosa Rhomboid. NRho consists of a novel alpha/beta fold and represents the first detailed structural insight into this class of intramembrane proteases. We find evidence that NRho is capable of strong and specific association with detergent micelles that mimic the membrane/water interface. Relaxation measurements on NRho reveal structural fluctuations on the microseconds-milliseconds timescale in regions including and contiguous to those implicated in membrane interaction. This structural plasticity may facilitate the ability of NRho to recognize and associate with membranes. We suggest that NRho plays a role in scissile peptide bond selectivity by optimally positioning the Rhomboid active site relative to the membrane plane.     

Structural Reorganization of α-Synuclein at Low pH Observed by NMR and REMD Simulations

α-Synuclein is an intrinsically disordered protein that appears in aggregated forms in the brains of patients with Parkinson's disease. The conversion from monomer to aggregate is complex, and aggregation rates are sensitive to changes in amino acid sequence and environmental conditions. It has previously been observed that α-synuclein aggregates faster at low pH than at neutral pH. Here, we combine NMR spectroscopy and molecular simulations to characterize α-synuclein conformational ensembles at both neutral and low pH in order to understand how the altered charge distribution at low pH changes the structural properties of these ensembles and leads to an increase in aggregation rate. The N-terminus, which has a small positive charge at neutral pH due to a balance of positively and negatively charged amino acid residues, is very positively charged at low pH. Conversely, the acidic C-terminus is highly negatively charged at neutral pH and becomes essentially neutral and hydrophobic at low pH. Our NMR experiments and replica exchange molecular dynamics simulations indicate that there is a significant structural reorganization within the low-pH ensemble relative to that at neutral pH in terms of long-range contacts, hydrodynamic radius, and the amount of heterogeneity within the conformational ensembles. At neutral pH, there is a very heterogeneous ensemble with transient contacts between the N-terminus and the non-amyloid β component (NAC); however, at low pH, there is a more homogeneous ensemble that exhibits strong contacts between the NAC and the C-terminus. At both pH values, transient contacts between the N- and C-termini are observed, the NAC region shows similar exposure to solvent, and the entire protein shows similar propensities to secondary structure. Based on the comparison of the neutral- and low-pH conformational ensembles, we propose that exposure of the NAC region to solvent and the secondary-structure propensity are not factors that account for differences in propensity to aggregate in this context. Instead, the comparison of the neutral- and low-pH ensembles suggests that the change in long-range interactions between the low- and neutral-pH ensembles, the compaction of the C-terminal region at low pH, and the uneven distribution of charges across the sequence are key to faster aggregation.     

Structure of the BH3 domains from the p53-inducible BH3-only proteins Noxa and Puma in complex with Mcl-1

Pro-survival proteins in the B-cell lymphoma-2 (Bcl-2) family have a defined specificity profile for their cell death-inducing BH3-only antagonists. Solution structures of myeloid cell leukaemia-1 (Mcl-1) in complex with the BH3 domains from Noxa and Puma, two proteins regulated by the tumour suppressor p53, show that they bind as amphipathic α-helices in the same hydrophobic groove of Mcl-1, using conserved residues for binding. Thermodynamic parameters for the interaction of Noxa, Puma and the related BH3 domains of Bmf, Bim, Bid and Bak with Mcl-1 were determined by calorimetry. These unstructured BH3 domains bind Mcl-1 with affinities that span 3 orders of magnitude, and binding is an enthalpically driven and entropy-enthalpy-compensated process. Alanine scanning analysis of Noxa demonstrated that only a subset of residues is required for interaction with Mcl-1, and these residues are localised to a short highly conserved sequence motif that defines the BH3 domain. Chemical shift mapping of Mcl-1:BH3 complexes showed that Mcl-1 engages all BH3 ligands in a similar way and that, in addition to changes in the immediate vicinity of the binding site, small molecule-wide structural adjustments accommodate ligand binding. Our studies show that unstructured peptides, such as the BH3 domains, behave like their structured counterparts and can bind tightly and selectively in an enthalpically driven process.     

Solution Structure and Phospholipid Interactions of the Isolated Voltage-Sensor Domain from KvAP

Voltage-sensor domains (VSDs) are specialized transmembrane segments that confer voltage sensitivity to many proteins such as ion channels and enzymes. The activities of these domains are highly dependent on both the chemical properties and the physical properties of the surrounding membrane environment. To learn about VSD-lipid interactions, we used nuclear magnetic resonance spectroscopy to determine the structure and phospholipid interface of the VSD from the voltage-dependent K⁺ channel KvAP (prokaryotic Kv from Aeropyrum pernix). The solution structure of the KvAP VSD solubilized within phospholipid micelles is similar to a previously determined crystal structure solubilized by a nonionic detergent and complexed with an antibody fragment. The differences observed include a previously unidentified short amphipathic α-helix that precedes the first transmembrane helix and a subtle rigid-body repositioning of the S3-S4 voltage-sensor paddle. Using ¹⁵N relaxation experiments, we show that much of the VSD, including the pronounced kink in S3 and the S3-S4 paddle, is relatively rigid on the picosecond-to-nanosecond timescale. In contrast, the kink in S3 is mobile on the microsecond-to-millisecond timescale and may act as a hinge in the movement of the paddle during channel gating. We characterized the VSD-phospholipid micelle interactions using nuclear Overhauser effect spectroscopy and showed that the micelle uniformly coats the KvAP VSD and approximates the chemical environment of a phospholipid bilayer. Using paramagnetically labeled phospholipids, we show that bilayer-forming lipids interact with the S3 and S4 helices more strongly than with S1 and S2.     

Restriction endonuclease inhibitor IPI* of bacteriophage T4: a novel structure for a dedicated target

Phage T4 protects its DNA from the two-gene-encoded gmrS/gmrD (glucose-modified hydroxymethylcytosine restriction endonuclease) CT of pathogenic Escherichia coli, CT596, by injecting several hundred copies of the 76-amino-acid-residue nuclease inhibitor, IPI*, into the infected host. Here, the three-dimensional solution structure of mature IPI* is reported as determined by nuclear magnetic resonance techniques using 1290 experimental nuclear Overhauser effect and dipolar coupling constraints (∼ 17 constraints per residue). Close examination of this oblate-shaped protein structure reveals a novel fold consisting of two small β-sheets (β1: B1 and B2; β2: B3-B5) flanked at the N- and C-termini by α-helices (H1 and H2). Such a fold is very compact in shape and allows ejection of IPI* through the narrow 30-Å portal and tail tube apertures of the virion without unfolding. Structural and dynamic measurements identify an exposed hydrophobic knob that is a putative gmrS/gmrD-binding site. A single gene from the uropathogenic E. coli UT189, which codes for a gmrS/gmrD-like UT fusion enzyme (with ∼ 90% identity to the heterodimeric CT enzyme), has evolved IPI* inhibitor immunity. Analysis of the gmrS/gmrD restriction endonuclease enzyme family and its IPI* family phage antagonists reveals an evolutionary pathway that has elaborated a surprisingly diverse and specifically fitted set of coevolving attack and defense structures.     

Solution NMR structure of putidaredoxin-cytochrome P450cam complex via a combined residual dipolar coupling-spin labeling approach suggests a role for Trp106 of putidaredoxin in complex formation

The 58-kDa complex formed between the [2Fe-2S] ferredoxin, putidaredoxin (Pdx), and cytochrome P450cam (CYP101) from the bacterium Pseudomonas putida has been investigated by high-resolution solution NMR spectroscopy. Pdx serves as both the physiological reductant and effector for CYP101 in the enzymatic reaction involving conversion of substrate camphor to 5-exo-hydroxycamphor. In order to obtain an experimental structure for the oxidized Pdx-CYP101 complex, a combined approach using orientational data on the two proteins derived from residual dipolar couplings and distance restraints from site-specific spin labeling of Pdx has been applied. Spectral changes for residues in and near the paramagnetic metal cluster region of Pdx in complex with CYP101 have also been mapped for the first time using ¹⁵N and ¹³C NMR spectroscopy, leading to direct identification of the residues strongly affected by CYP101 binding. The new NMR structure of the Pdx-CYP101 complex agrees well with results from previous mutagenesis and biophysical studies involving residues at the binding interface such as formation of a salt bridge between Asp38 of Pdx and Arg112 of CYP101, while at the same time identifying key features different from those of earlier modeling studies. Analysis of the binding interface of the complex reveals that the side chain of Trp106, the C-terminal residue of Pdx and critical for binding to CYP101, is located across from the heme-binding loop of CYP101 and forms non-polar contacts with several residues in the vicinity of the heme group on CYP101, pointing to a potentially important role in complex formation.     

An Extended Structure of the APOBEC3G Catalytic Domain Suggests a Unique Holoenzyme Model

Human APOBEC3G (A3G) belongs to a family of polynucleotide cytidine deaminases. This family includes APOBEC1 and AID, which edit APOB mRNA and antibody gene DNA, respectively. A3G deaminates cytidines to uridines in single-strand DNA and inhibits the replication of human immunodeficiency virus-1, other retroviruses, and retrotransposons. Although the mechanism of A3G-catalyzed DNA deamination has been investigated genetically and biochemically, atomic details are just starting to emerge. Here, we compare the DNA cytidine deaminase activities and NMR structures of two A3G catalytic domain constructs. The longer A3G191-384 protein is considerably more active than the shorter A3G198-384 variant. The longer structure has an α1-helix (residues 201-206) that was not apparent in the shorter protein, and it contributes to catalytic activity through interactions with hydrophobic core structures (β1, β3, α5, and α6). Both A3G catalytic domain solution structures have a discontinuous β2 region that is clearly different from the continuous β2 strand of another family member, APOBEC2. In addition, the longer A3G191-384 structure revealed part of the N-terminal pseudo-catalytic domain, including the interdomain linker and some of the last α-helix. These structured residues (residues 191-196) enabled a novel full-length A3G model by providing physical overlap between the N-terminal pseudo-catalytic domain and the new C-terminal catalytic domain structure. Contrary to predictions, this structurally constrained model suggested that the two domains are tethered by structured residues and that the N- and C-terminal β2 regions are too distant from each other to participate in this interaction.     

Characterization of conformational and dynamic properties of natively unfolded human and mouse alpha-synuclein ensembles by NMR: implication for aggregation

Conversion of human α-synuclein (aS) from the free soluble state to the insoluble fibrillar state has been implicated in the etiology of Parkinson's disease. Human aS is highly homologous in amino acid sequence to mouse aS, which contains seven substitutions including the A53T that has been linked to familial Parkinson's disease, and including five substitutions in the C-terminal region. It has been shown that the rate of fibrillation is highly dependent on the exact sequence of the protein, and mouse aS is reported to aggregate more rapidly than human aS in vitro. Nuclear magnetic resonance experiments of mouse and human aS at supercooled temperatures (263 K) are used to understand the effect of sequence on conformational fluctuations in the disordered ensembles and to relate these to differences in propensities to aggregate. We show that both aS are natively unfolded at low temperature with different propensities to secondary structure, backbone dynamics and long-range contacts across the protein. Mouse aS exhibits a higher propensity to helical conformation around the C-terminal substitutions as well as the loss of transient long-range contacts from the C- to the N-terminal end and hydrophobic central regions of the protein relative to human aS. Lack of back-folding from the C-terminal end of mouse aS exposes the N-terminal region, which is shown, by ¹⁵N relaxation experiments, to be very restricted in mobility relative to human aS. We propose that the restricted mobility in the N-terminal region may arise from transient interchain interactions, suggesting that the N-terminal KTK(E/Q)GV repeats may serve as initiation sites for aggregation in mouse aS. These transient interchain interactions coupled with a non-Aβ amyloid component (NAC) region that is both more exposed and has a higher propensity to β structure may accelerate the rate of fibril formation of aS.     

Mis-translation of a computationally designed protein yields an exceptionally stable homodimer: implications for protein engineering and evolution

We recently used computational protein design to create an extremely stable, globular protein, Top7, with a sequence and fold not observed previously in nature. Since Top7 was created in the absence of genetic selection, it provides a rare opportunity to investigate aspects of the cellular protein production and surveillance machinery that are subject to natural selection. Here we show that a portion of the Top7 protein corresponding to the final 49 C-terminal residues is efficiently mis-translated and accumulates at high levels in Escherichia coli. We used circular dichroism, size-exclusion chromatography, small-angle X-ray scattering, analytical ultra-centrifugation, and NMR spectroscopy to show that the resulting C-terminal fragment (CFr) protein adopts a compact, extremely stable, homo-dimeric structure. Based on the solution structure, we engineered an even more stable variant of CFr by disulfide-induced covalent circularisation that should be an excellent platform for design of novel functions. The accumulation of high levels of CFr exposes the high error rate of the protein translation machinery. The rarity of correspondingly stable fragments in natural proteins coupled with the observation that high quality ribosome binding sites are found to occur within E. coli protein-coding regions significantly less often than expected by random chance implies a stringent evolutionary pressure against protein sub-fragments that can independently fold into stable structures. The symmetric self-association between two identical mis-translated CFr sub-domains to generate an extremely stable structure parallels a mechanism for natural protein-fold evolution by modular recombination of protein sub-structures.