The C-terminus of dUTPase: observation on flexibility using NMR

The dynamics of the C-terminus of the dUTPases from Escherichia coli and equine infectious anaemia virus (EIAV) were studied by 1H-(15)N nuclear magnetic resonance spectroscopy. The two enzymes differ with regard to flexibility in the backbone of the 15 most C-terminal amino acid residues, some of which are conserved and essential for enzymic activity. In the bacterial enzyme, the residues closest to the C-terminus are highly flexible and display a correlation time in the nanosecond time range. No similar high flexibility could be detected for the C-terminal part of EIAV dUTPase, indicating a different time range of flexibility.     

Nonnative helical motif in a chaperone-bound protein fragment

The effect of cotranslationally active chaperones on the conformation of incomplete protein chains is poorly understood. The secondary structure of a 77-residue chaperone-bound N-terminal protein fragment corresponding to the first five helices (A-E) of apomyoglobin (apoMb_1-77) is investigated here at the residue-specific level by multidimensional NMR. The substrate-binding domain of DnaK, DnaK-β, is employed as a chaperone model. By taking advantage of the improved spectral quality resulting from chaperone deuteration, we find that DnaK-β-bound apoMb_1-77 displays a region of nonnative helicity at residues away from the main chaperone binding site. The nonnative structural motif comprises portions of the native D and E helices and has similar characteristics to the reported nonnative DE helical region of acid-unfolded full-length apoMb. Upon incorporation of the missing C-terminal amino acids, a structural kink develops between residues 56 and 57, and two separate native D and E helices are generated. This work highlights, for the first time to our knowledge, the presence of a nonnative helical motif in a large chaperone-bound protein fragment under physiologically relevant solution conditions.     

Crystal Structure of a Four-Layer Aggregate of Engineered TMV CP Implies the Importance of Terminal Residues for Oligomer Assembly

Background

Crystal structures of the tobacco mosaic virus (TMV) coat protein (CP) in its helical and disk conformations have previously been determined at the atomic level. For the helical structure, interactions of proteins and nucleic acids in the main chains were clearly observed; however, the conformation of residues at the C-terminus was flexible and disordered. For the four-layer aggregate disk structure, interactions of the main chain residues could only be observed through water–mediated hydrogen bonding with protein residues. In this study, the effects of the C-terminal peptides on the interactions of TMV CP were investigated by crystal structure determination.

Methodology/Principal Findings

The crystal structure of a genetically engineered TMV CP was resolved at 3.06 Å. For the genetically engineered TMV CP, a six-histidine (His) tag was introduced at the N-terminus, and the C-terminal residues 155 to 158 were truncated (N-His-TMV CP¹⁹). Overall, N-His-TMV CP¹⁹ protein self-assembled into the four-layer aggregate form. The conformations of residues Gln36, Thr59, Asp115 and Arg134 were carefully analyzed in the high radius and low radius regions of N-His-TMV CP¹⁹, which were found to be significantly different from those observed previously for the helical and four-layer aggregate forms. In addition, the aggregation of the N-His-TMV CP¹⁹ layers was found to primarily be mediated through direct hydrogen-bonding. Notably, this engineered protein also can package RNA effectively and assemble into an infectious virus particle.

Conclusion

The terminal sequence of amino acids influences the conformation and interactions of the four-layer aggregate. Direct protein–protein interactions are observed in the major overlap region when residues Gly155 to Thr158 at the C-terminus are truncated. This engineered TMV CP is reassembled by direct protein–protein interaction and maintains the normal function of the four-layer aggregate of TMV CP in the presence of RNA.     

Solution structure and mapping of a very weak calcium-binding site of human translationally controlled tumor protein by NMR

Human translationally controlled tumor protein (TCTP) is a growth-related, calcium-binding protein. We determined the solution structure and backbone dynamics of human TCTP, and identified the calcium-binding site of human TCTP using multi-dimensional NMR spectroscopy. The overall structure of human TCTP has a rather rigid well-folded core and a very flexible long loop connected by a short two-strand β-sheet, which shows a conserved fold in the TCTP family. The C-terminal portions of loop L_α3β8 and strand β9 and the N-terminal region of strand β8 may form a calcium-binding site in the human TCTP structure, which is largely conserved in the sequence alignment of TCTPs. The K_d value for the calcium binding is 0.022-0.025 M indicating a very weak calcium-binding site.     

NMR dynamics-derived insights into the binding properties of a peptide interacting with an SH2 domain

The signal transduction protein phospholipase C-gamma1 (PLC-gamma1) is activated when its C-terminal SH2 domain (PLCC) binds the phosphorylated Tyr-1021 site (pTyr-1021) in the beta-platelet-derived growth factor receptor (PDGFR). To better understand the contributions that dynamics make to binding, we have used NMR relaxation experiments to investigate the motional properties of backbone amide and side chain methyl groups in a peptide derived from the pTyr-1021 site of PDGFR, both free and in complex with the PLCC SH2 domain. The free peptide has relaxation properties that are typical for a small, unstructured polymer, while the backbone of the bound peptide is least flexible for residues in the central portion of the binding site with the amplitude of pico- to nanosecond time scale motions increasing toward the C-terminus of the peptide. The increase in large amplitude motion toward the end of the pY1021 peptide is consistent with the bound peptide existing as an ensemble of states with C-terminal residues having the broadest distribution of backbone conformations, while residues in the central binding site are the most restricted. Deuterium spin relaxation experiments establish that the protein-peptide interface is highly dynamic, and this mobility may play an important role in modulating the affinity of the interaction.     

15N magnetic relaxation study of backbone dynamics of the ribosome-associated cold shock response protein Yfia of Escherichia coli

In the solution structure of the ribosome-associated cold shock response protein Yfia of Escherichia coli in the free state two structural segments can be distinguished: a well structured, rigid N-terminal part displaying a betaalphabetabetabetaalpha topology and a flexible C-terminal tail comprising last 20 amino-acid residues. The backbone dynamics of Yfia protein was studied by (15)N nuclear magnetic relaxation at three magnetic fields and analyzed using model-free approach. The overall diffusional tumbling of the N-terminal part is strongly anisotropic with a number of short stretches showing increased mobility either on a subnanosecond time scale, or a micro- to millisecond time scale, or both. In contrast, the unstructured polypeptide chain of the C-terminal part, which cannot be regarded as a rigid structure, shows the predominance of fast local motions over slower ones, both becoming faster closer to the C-terminus.     

Disorder in the Human Skp1 Structure is the Key to its Adaptability to Bind Many Different Proteins in the SCF Complex Assembly

Skp1(S-phase kinase-associated protein 1 - Homo sapiens) is an adapter protein of the SCF(Skp1-Cullin1-Fbox) complex, which links the constant components (Cul1-RBX) and the variable receptor (F-box proteins) in Ubiquitin E3 ligase. It is intriguing how Skp1 can recognise and bind to a variety of structurally different F-box proteins. For practical reasons, previous efforts have used truncated Skp1, and thus it has not been possible to track the crucial aspects of the substrate recognition process. In this background, we report the solution structure of the full-length Skp1 protein determined by NMR spectroscopy for the first time and investigate the sequence-dependent dynamics in the protein. The solution structure reveals that Skp1 has an architecture: β1-β2-H1-H2-L1–H3-L2-H4-H5-H6-H7(partially formed) and a long tail-like disordered C-terminus. Structural analysis using DALI (Distance Matrix Alignment) reveals conserved domain structure across species for Skp1. Backbone dynamics investigated using NMR relaxation suggest substantial variation in the motional timescales along the length of the protein. The loops and the C-terminal residues are highly flexible, and the (R₂/R₁) data suggests μs-ms timescale motions in the helices as well. Further, the dependence of amide proton chemical shift on temperature and curved profiles of their residuals indicate that the residues undergo transitions between native state and excited state. The curved profiles for several residues across the length of the protein suggest that there are native-like low-lying excited states, particularly for several C-terminal residues. Our results provide a rationale for how the protein can adapt itself, bind, and get functionally associated with other proteins in the SCF complex by utilising its flexibility and conformational sub-states.     

The structure and orientation of the C-terminus of LRAP

Amelogenin is the predominant protein found during enamel development and is thought to be the biomineralization protein controlling the unique elongated hydroxyapatite crystals that constitute enamel. The secondary structure of biomineralization proteins is thought to be important in the interaction with hydroxyapatite. Unfortunately, very little data are available on the structure or the orientation of amelogenin, either in solution or bound to hydroxyapatite. The C-terminus contains the majority of the charged residues and is predicted to interact with hydroxyapatite; thus, we used solid-state NMR dipolar recoupling techniques to investigate the structure and orientation of the C-terminus of LRAP, a naturally occurring splice variant of full-length amelogenin. Using ¹³C{¹⁵N} Rotational Echo DOuble Resonance (REDOR), the structure of the C-terminus was found to be largely random coil, both on the surface of hydroxyapatite as well as lyophilized from solution. The orientation of the C-terminal region with respect to hydroxyapatite was investigated for two alanine residues (Ala⁴⁶ and Ala⁴⁹) using ¹³C{³¹P} REDOR and one lysine residue (Lys⁵²) using ¹⁵N{³¹P} REDOR. The residues examined were found to be 7.0, 5.7, and 5.8 Å from the surface of hydroxyapatite for Ala⁴⁶, Ala⁴⁹, and Lys⁵², respectively. This provides direct evidence that the charged C-terminus is interacting closely with hydroxyapatite, positioning the acidic amino acids to aid in controlling crystal growth. However, solid-state NMR dynamics measurements also revealed significant mobility in the C-terminal region of the protein, in both the side chains and the backbone, suggesting that this region alone is not responsible for binding.     

Impact of N-glycosylation site variants during human PrP aggregation and fibril nucleation

Misfolding and aggregation of the human prion protein (PrP) cause neurodegenerative transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease. Mature native PrP is composed of 209 residues and is folded into a C-terminal globular domain (residues 125–209) comprising a small two-stranded β-sheet and three α-helices. The N-terminal domain (residues 23–124) is intrinsically disordered. Expression of truncated PrP (residues 90–231) is sufficient to cause prion disease and residues 90/100–231 is comprising the amyloid-like fibril core of misfolded infectious PrP. During PrP fibril formation under native conditions in vitro, the disordered N-terminal domain slows down fibril formation likely due to a mechanism of initial aggregation forming morphologically disordered aggregates. The morphological disordered aggregate is a transient phase. Nucleation of fibrils occurs from this initial aggregate. The aggregate phase is largely circumvented by seeding with preformed PrP fibrils. In vivo PrP is N-glycosylated at positions Asn181 and Asn197. Little is known about the importance of these positions and their glycans for PrP stability, aggregation and fibril formation. We have in this study taken a step towards that goal by mutating residues 181 and 197 for cysteines to study the positional impact on these processes. We have further by organic synthetic chemistry and chemical modification generated synthetic glycosylations in these positions. Our data shows that residue 181 when mutated to a cysteine is a key residue for self-chaperoning, rendering a trap in the initial aggregate preventing conformational changes towards amyloid fibril formation. Position 197 is less involved in the aggregate trapping and is more geared towards β-sheet structure conversion within amyloid fibrils. As expected, synthetic glycosylated 197 is less affected towards fibril formation compared to glycosylated 181. Our data are rather compatible with the parallel in-register intermolecular β-sheet model structure of the PrP90–231 fibril and sheds light on the misfolding transitions of PrP in vitro. We hypothesize that glycosylation of position 181 is a key site for prion strain differentiation in vivo.     

Aggregation kinetics of interrupted polyglutamine peptides

Abnormally expanded polyglutamine domains are associated with at least nine neurodegenerative diseases, including Huntington's disease. Expansion of the glutamine region facilitates aggregation of the impacted protein, and aggregation has been linked to neurotoxicity. Studies of synthetic peptides have contributed substantially to our understanding of the mechanism of aggregation because the underlying biophysics of polyglutamine-mediated association can be probed independent of their context within a larger protein. In this report, interrupting residues were inserted into polyglutamine peptides (Q20), and the impact on conformational and aggregation properties was examined. A peptide with two alanine residues formed laterally aligned fibrillar aggregates that were similar to the uninterrupted Q20 peptide. Insertion of two proline residues resulted in soluble, nonfibrillar aggregates, which did not mature into insoluble aggregates. In contrast, insertion of a β-turn template _DPG rapidly accelerated aggregation and resulted in a fibrillar aggregate morphology with little lateral alignment between fibrils. These results are interpreted to indicate that (a) long-range nonspecific interactions lead to the formation of soluble oligomers, while maturation of oligomers into fibrils requires conformational conversion and (b) that soluble oligomers dynamically interact with each other, while insoluble aggregates are relatively inert. Kinetic analysis revealed that the increase in aggregation caused by the _DPG insert is inconsistent with the nucleation-elongation mechanism of aggregation featuring a monomeric β-sheet nucleus. Rather, the data support a mechanism of polyglutamine aggregation by which monomers associate into soluble oligomers, which then undergo slow structural rearrangement to form sedimentable aggregates.     

Formation of soluble inclusion bodies by hpv e6 oncoprotein fused to maltose-binding protein

Many polypeptides overexpressed in bacteria are produced misfolded and accumulate as solid structures called inclusion bodies. Inclusion-body-prone proteins have often been reported to escape precipitation when fused to maltose-binding protein (MBP). Here, we have examined the case of HPV 16 oncoprotein E6. The unfused sequence of E6 is overexpressed as inclusion bodies in bacteria. By contrast, fusions of E6 to the C-terminus of MBP are produced soluble. We have analyzed preparations of soluble MBP-E6 fusions by using three independent approaches: dynamic light scattering, lateral turbidimetry, and sandwich ELISA. All three methods showed that MBP-E6 preparations contain highly aggregated material. The behavior of these soluble aggregates under denaturating conditions suggests that they are formed by agglomeration of misfolded E6 moieties. However, precipitation is prevented by the presence of the folded and highly soluble MBP moieties, which maintain the aggregates in solution. Therefore, the fact that a protein or protein domain is produced soluble when fused to the C-terminus of a carrier protein does not guarantee that the protein of interest is properly folded and active. We suggest that aggregation of fusion proteins should be systematically assayed, especially when these fusions are to be used for binding measurements or activity tests.     

Molecular dynamics simulations of two tandem octarepeats from the mammalian prion protein: fully Cu2+-bound and metal-free forms

Molecular dynamics simulations have been conducted on a model fragment (Ac-PHGGGWGQPHGGGW-NH₂) of the prion protein octarepeat domain, both in the Cu²⁺-bound and metal-free forms. The copper-bound models are based on the consensus structure of the core Cu²⁺-binding site of an individual octarepeat, relevant to the fully Cu²⁺-occupied prion protein octarepeat region. The model peptides contain Cu²⁺ bound through a His imidazole ring and two deprotonated amide N-atoms in the peptide backbone supplied by the following two Gly residues. Both the copper-bound and metal-free models have been simulated with the OPLS all-atom force field with the GROMACS molecular dynamics package. These simulations, with two tandem copper-binding sites, represent the minimum model necessary to observe potential structuring between the copper-binding sites in the octarepeat region. The GWGQ residues constitute a flexible linker region that predominantly adopts a turn, serving to bring adjacent His residues into close proximity. The consequent formation of stable structures demonstrates that the copper-bound octarepeat region allows the copper-coordinating sites to come into van der Waals contact, packing into particular orientations to further stabilize the bend in the GWGQ linker region.     

Can copper binding to the prion protein generate a misfolded form of the protein?

The native prion protein (PrP) has a two domain structure, with a globular folded α-helical C-terminal domain and a flexible extended N-terminal region. The latter can selectively bind Cu²⁺ via four His residues in the octarepeat (OR) region, as well as two sites (His96 and His111) outside this region. In the disease state, the folded C-terminal domain of PrP undergoes a conformational change, forming amorphous aggregates high in β-sheet content. Cu²⁺ bound to the ORs can be redox active and has been shown to induce cleavage within the OR region, a process requiring conserved Trp residues. Using computational modeling, we have observed that electron transfer from Trp residues to copper can be favorable. These models also reveal that an indole-based radical cation or Cu⁺ can initiate reactions leading to protein backbone cleavage. We have also demonstrated, by molecular dynamics simulations, that Cu²⁺ binding to the His96 and His111 residues in the remaining PrP N-terminal fragment can induce localized β-sheet structure, allowing us to suggest a potential mechanism for the initiation of β-sheet misfolding in the C-terminal domain by Cu²⁺.

Protein mutagenesis with monodispersity-based quality probing: selective inactivation of p53 degradation and DNA-binding properties of HPV E6 oncoprotein

Interpretation of protein mutagenesis experiments requires the ability to distinguish functionally relevant mutations from mutations affecting the structure. When a protein is expressed soluble in bacteria, properly folded mutants are expected to remain soluble whereas misfolded mutants should form insoluble aggregates. However, this rule may fail for proteins fused to highly soluble carrier proteins. In a previous study, we analysed the biophysical status of HPV oncoprotein E6 fused to the C-terminus of maltose-binding protein (MBP) and found that misfolded E6 moieties fused to MBP formed soluble aggregates of high molecular weight. By contrast, preparations of properly folded E6 fused to MBP were monodisperse. Here, we have used this finding to evaluate the quality of 19 MBP-fused E6 site-directed mutants by using a light scattering assay performed in a fluorimeter. This assay guided us to rule out structurally defective mutants and to obtain functionally relevant E6 mutants selectively altered for two molecular activities: degradation of tumour suppressor p53 and DNA recognition.     

Insight into Early-Stage Unfolding of GPI-Anchored Human Prion Protein

Prion diseases are fatal neurodegenerative disorders, which are characterized by the accumulation of misfolded prion protein (PrP^Sc) converted from a normal host cellular prion protein (PrP^C). Experimental studies suggest that PrP^C is enriched with α-helical structure, whereas PrP^Sc contains a high proportion of β-sheet. In this study, we report the impact of N-glycosylation and the membrane on the secondary structure stability utilizing extensive microsecond molecular dynamics simulations. Our results reveal that the HB (residues 173 to 194) C-terminal fragment undergoes conformational changes and helix unfolding in the absence of membrane environments because of the competition between protein backbone intramolecular and protein-water intermolecular hydrogen bonds as well as its intrinsic instability originated from the amino acid sequence. This initiation of the unfolding process of PrP^C leads to a subsequent increase in the length of the HB-HC loop (residues 195 to 199) that may trigger larger rigid body motions or further unfolding around this region. Continuous interactions between prion protein and the membrane not only constrain the protein conformation but also decrease the solvent accessibility of the backbone atoms, thereby stabilizing the secondary structure, which is enhanced by N-glycosylation via additional interactions between the N-glycans and the membrane surface.     

Autophagy and proteotoxicity in cardiomyocytes

Increasing evidence suggests that misfolded proteins and intracellular aggregates contribute to cardiac disease and heart failure. We wished to determine if autophagic induction by Atg7 is sufficient to reduce misfolded protein and aggregate content in protein misfolding-stressed cardiomyocytes. We used loss- and gain-of-function approaches in cultured cardiomyocytes to determine the effects of ATG7 knockdown and Atg7 overexpression in protein conformation-based toxicity induced by expression of a mutant aB crystallin (CryAB^R120G) known to cause human heart disease. We show that Atg7 induces basal autophagy and rescues the CryAB accumulation of misfolded proteins and aggregates in cardiomyocytes.     

The distribution of residues in a polypeptide sequence is a determinant of aggregation optimized by evolution

It has been shown that the propensity of a protein to form amyloid-like fibrils can be predicted with high accuracy from the knowledge of its amino acid sequence. It has also been suggested, however, that some regions of the sequences are more important than others in determining the aggregation process. Here, we have addressed this issue by constructing a set of “sequence scrambled” variants of the first 29 residues of horse heart apomyoglobin (apoMb_1-29), in which the sequence was modified while maintaining the same amino acid composition. The clustering of the most amyloidogenic residues in one region of the sequence was found to cause a marked increase of the elongation rate (k_agg) and a remarkable shortening of the lag phase (t_lag) of the fibril growth, as determined by far-UV circular dichroism and thioflavin T fluorescence. We also show that taking explicitly into consideration the presence of aggregation-promoting regions in the predictive methods results in a quantitative agreement between the theoretical and observed k_agg and t_lag values of the apoMb_1-29 variants. These results, together with a comparison between homologous segments from the family of globins, indicate the existence of a negative selection against the clustering of highly amyloidogenic residues in one or few regions of polypeptide sequences.     

Disulfide linkage in the coiled-coil domain of subunit H of A1AO ATP synthase from Methanocaldococcus jannaschii and the NMR structure of the C-terminal segment H85-104

The C-terminal residues 98-104 are important for structure stability of subunit H of A₁A_O ATP synthases as well as its interaction with subunit A. Here we determined the structure of the segment H_85-104 of H from Methanocaldococcus jannaschii, showing a helix between residues Lys90 to Glu100 and flexible tails at both ends. The helix-helix arrangement in the C-terminus was investigated by exchange of hydrophobic residues to single cysteine in mutants of the entire subunit H (H_I93C, H_L96C and H_L98C). Together with the surface charge distribution of H_85-104, these results shine light into the A-H assembly of this enzyme.     

Characterisation of amyloid fibril formation by small heat-shock chaperone proteins human alphaA-, alphaB- and R120G alphaB-crystallins

AlphaB-Crystallin is a ubiquitous small heat-shock protein (sHsp) renowned for its chaperone ability to prevent target protein aggregation. It is stress-inducible and its up-regulation is associated with a number of disorders, including those linked to the deposition of misfolded proteins, such as Alzheimer's and Parkinson's diseases. We have characterised the formation of amyloid fibrils by human alphaB-crystallin in detail, and also that of alphaA-crystallin and the disease-related mutant R120G alphaB-crystallin. We find that the last 12 amino acid residues of the C-terminal region of alphaB-crystallin are predicted from their physico-chemical properties to have a very low propensity to aggregate. (1)H NMR spectroscopy reveals that this hydrophilic C-terminal region is flexible both in its solution state and in amyloid fibrils, where it protrudes from the fibrillar core. We demonstrate, in addition, that the equilibrium between different protofilament assemblies can be manipulated and controlled in vitro to select for particular alphaB-crystallin amyloid morphologies. Overall, this study suggests that there could be a fine balance in vivo between the native functional sHsp state and the formation of amyloid fibrils.