Characterization of the nucleation step and folding of a collagen triple-helix peptide

Peptide T1-892 is a triple-helical peptide designed to include two distinct domains: a C-terminal (Gly-Pro-Hyp)(4) sequence, together with an N-terminal 18-residue sequence from the alpha1(I) chain of type I collagen. Folding experiments of T1-892 using CD spectroscopy were carried out at varying concentrations and temperatures, and fitting of kinetic models to the data was used to obtain information about the folding mechanism and to derive rate constants. Proposed models include a heterogeneous population of monomers with respect to cis-trans isomerization and a third-order folding reaction from competent monomer to the triple helix. Fitting results support a nucleation domain composed of all or most of the (Gly-Pro-Hyp)(4) sequence, which must be in trans form before the monomer is competent to initiate triple-helix formation. The folding of competent monomer to a triple helix is best described by an all-or-none third-order reaction. The temperature dependence of the third-order rate constant indicates a negative activation energy and provides information about the thermodynamics of the trimerization step. These CD studies complement NMR studies carried out on the same peptide at high concentrations, illustrating how the rate-limiting folding step is affected by changes in concentration. This sequence preference of repeating Gly-Pro-Hyp units for the initiation of triple-helix formation in peptide T1-892 may be related to features in the triple-helix folding of collagens.     

Location of glycine mutations within a bacterial collagen protein affects degree of disruption of triple-helix folding and conformation

The hereditary bone disorder osteogenesis imperfecta is often caused by missense mutations in type I collagen that change one Gly residue to a larger residue and that break the typical (Gly-Xaa-Yaa)(n) sequence pattern. Site-directed mutagenesis in a recombinant bacterial collagen system was used to explore the effects of the Gly mutation position and of the identity of the residue replacing Gly in a homogeneous collagen molecular population. Homotrimeric bacterial collagen proteins with a Gly-to-Arg or Gly-to-Ser replacement formed stable triple-helix molecules with a reproducible 2 °C decrease in stability. All Gly replacements led to a significant delay in triple-helix folding, but a more dramatic delay was observed when the mutation was located near the N terminus of the triple-helix domain. This highly disruptive mutation, close to the globular N-terminal trimerization domain where folding is initiated, is likely to interfere with triple-helix nucleation. A positional effect of mutations was also suggested by trypsin sensitivity for a Gly-to-Arg replacement close to the triple-helix N terminus but not for the same replacement near the center of the molecule. The significant impact of the location of a mutation on triple-helix folding and conformation could relate to the severe consequences of mutations located near the C terminus of type I and type III collagens, where trimerization occurs and triple-helix folding is initiated.     

Schmid's metaphyseal chondrodysplasia mutations interfere with folding of the C-terminal domain of human collagen X expressed in Escherichia coli.

Human collagen X contains a highly conserved 161-amino acid C-terminal non-triple helical domain that is homologous to the C-terminal domain of collagen VIII and to the C1q module of the human C1 enzyme. We have expressed this domain (residues 545-680) in Escherichia coli as a glutathione S-transferase fusion protein. The purified fusion protein trimerizes spontaneously in vitro, and after thrombin cleavage, the purified C-terminal domain trimer (46.2 kDa) is extremely stable and trypsin-resistant. Mutations within the C-terminal domain have been observed in patients with Schmid's metaphyseal chondrodysplasia (SMCD). Some of these mutations (Y598D, G618V, W651X, or H669X; X is the stop codon) were constructed by site-directed mutagenesis. Each mutation had identical consequences regarding the fusion protein: 1) absence of trimeric formation, 2) copurification of the approximately 60-kDa GroEL chaperone protein, and 3) sensitivity of the monomeric fusion protein to trypsin digestion. These results show that the C-terminal domain of collagen X is sufficient to produce a very stable and compact trimer in the absence of collagen Gly-X-Y repeats. Moreover, mutations causing SMCD interfere in this system with the correct folding of the C-terminal domain. The existence of a similar mechanism in chondrocytes might explain the relative homogeneity of phenotypes in SMCD despite the diversity of mutations.     

A peptide study of the relationship between the collagen triple-helix and amyloid

Type XXV collagen, or collagen‐like amyloidogenic component, is a component of amyloid plaques, and recent studies suggest this collagen affects amyloid fibril elongation and has a genetic association with Alzheimer's disease. The relationship between the collagen triple helix and amyloid fibrils was investigated by studying peptide models, including a very stable triple helical peptide (Pro‐Hyp‐Gly)₁₀, an amyloidogenic peptide GNNQQNY, and a hybrid peptide where the GNNQQNY sequence was incorporated between (GPO)_n domains. Circular dichroism and nuclear magnetic resonance (NMR) spectroscopy showed the GNNQQNY peptide formed a random coil structure, whereas the hybrid peptide contained a central disordered GNNQQNY region transitioning to triple‐helical ends. Light scattering confirmed the GNNQQNY peptide had a high propensity to form amyloid fibrils, whereas amyloidogenesis was delayed in the hybrid peptide. NMR data suggested the triple‐helix constraints on the GNNQQNY sequence within the hybrid peptide may disfavor the conformational change necessary for aggregation. Independent addition of a triple‐helical peptide to the GNNQQNY peptide under aggregating conditions delayed nucleation and amyloid fibril growth. The inhibition of amyloid nucleation depended on the Gly‐Xaa‐Yaa sequence and required the triple‐helix conformation. The inhibitory effect of the collagen triple‐helix on an amyloidogenic sequence, when in the same molecule or when added separately, suggests Type XXV collagen, and possibly other collagens, may play a role in regulating amyloid fibril formation. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 795–806, 2012.     

Presence of a thermostable domain in the helical part of the type I collagen molecule and its role in the mechanism of triple helix folding.

A study has been done of the effect of neutral salts (NaCl and CaCl2) on the mechanism of type I collagen triple helix folding and unfolding in concentrated acetic acid solutions (2-8.8 M). It is shown that in these conditions, thermoabsorption and secondary structure change in heated solutions proceed in two consecutive stages. Salts exert a different destabilizing effect on different sites of the macromolecule, promoting the detection of a thermostable domain. The presence of a thermostable domain permits one to carry out reversible denaturation of collagen and to study the mechanism of the triple helix folding. Proceeding from the mechanism of the triple helix folding, an assumption has been made on the localization of the thermostable domain and its biological role.     

Expression of an exogenous peptide epitope genetically engineered in the variable domain of an immunoglobulin: implications for antibody and peptide folding.

Immunoglobulins bind antigens and express individual antigenic specificities mainly through residues located in hypervariable loops of their N-terminal domains. Hypervariable loops are kept in place by a molecular scaffold organized in a sandwich-like structure with two beta-sheets stabilized by a disulfide bridge (the immunoglobulin fold). This structural feature, together with the possibility of obtaining high level expression, extracellular secretion, easy purification and stability of the protein product, render immunoglobulin an ideal 'molecular vehicle' for the expression of exogenous peptides. Here we report on the engineering of an immunoglobulin expressing an exogenous epitope, the repetitive tetrapeptide Asn-Ala-Asn-Pro (NANP)3. By recombinant DNA techniques, we inserted three copies of the tetrapeptide (NANP)3 in the third hypervariable loop (D region) of an immunoglobulin heavy chain variable domain. We show that the engineered antibody was properly assembled and secreted. A panel of polyclonal and monoclonal antibodies, including anti-synthetic peptides and anti-(NANP)n antibodies, were used to study the molecular configuration of the engineered domain's surface. The results indicate that (i) the exogenous sequence did not appreciably alter the overall fold of the variable domain; and (ii) the inserted epitope folded with a configuration immunologically similar to the one assumed in the native protein, suggesting that short- and medium- rather than long-range interactions stabilized the structure of the (NANP)3 peptide in the folded protein. We propose this system for the expression of peptidic sequences, and their structural and functional analysis.     

The C-terminal extrahelical peptide of type I collagen and its role in fibrillogenesis in vitro

The formation in vitro of fibrils from type I acid-soluble calf skin collagen has been studied before and after removal of the extrahelical peptides with carboxypeptidase and with pepsin. Turbidimetric studies show that the mechanism of fibril growth in undigested collagen is similar to that in pepsin-digested collagen; following carboxypeptidase digestion, however, a different growth mechanism was apparent. The two mechanisms have been further characterized by electron microscopy. In the course of formation of fibrils from undigested collagen, “early fibrils” (short D-periodic fibrils that have both ends visible) occurred in the lag phase under the precipitating conditions employed here. After pepsin or carboxypeptidase digestion of the collagen no “early fibrils” were seen. In carboxypeptidase-digested collagen, lateral assembly was inhibited; after pepsin digestion, linear assembly was inhibited. Complete removal of the extrahelical peptides prevented fibril formation under the conditions used here. Electron-optical examination of segment-long-spacing (SLS) dimers established a more complete removal of the C-terminal peptide after carboxypeptidase digestion than after pepsin digestion. Analyses of staining patterns of SLS dimers and fibrils from undigested and digested samples showed that the C-terminal peptide in SLS crystallites and fibrils formed from undigested collagen is in a condensed conformation. A proposed conformation, in which condensation occurs predominantly in a hydrophobic region at the proximal end of the C-terminal peptide, is discussed in terms of a dual role for the C-terminal peptide in fibrillogenesis. One role, shared with the N-terminal peptide, is to participate in interactions between the 4D-staggered molecules leading to the formation of linear aggregates; the other is to participate in interactions between these linear aggregates giving rise to D-periodic aggregates and lateral (as well as linear) growth.     

The folding mechanism of a beta-sheet: the WW domain

The folding thermodynamics and kinetics of the Pin WW domain, a three-stranded antiparallel beta-sheet, have been characterized extensively. Folding and activation free energies were determined as a function of temperature for 16 mutants, which sample all strands and turns of the molecule. The mutational phi value (Phi(m)) diagram is a smooth function of sequence, indicating a prevalence of local interactions in the transition state (TS). At 37 degrees C, the diagram has a single pronounced maximum at turn 1: the rate-limiting step during folding is the formation of loop 1. In contrast, key residues for thermodynamic stability are located in the strand hydrophobic clusters, indicating that factors contributing to protein stability and folding kinetics are not correlated. The location of the TS along the entropic reaction coordinate Phi(T), obtained by temperature-tuning the kinetics, reveals that sufficiently destabilizing mutants in loop 2 or in the Leu7-Trp11-Tyr24-Pro37 hydrophobic cluster can cause a switch to a late TS. Phi(m) analysis is usually applied "perturbatively" (methyl truncation), but with Phi(T) to quantitatively assess TS shifts along a reaction coordinate, more severe mutations can be used to probe regions of the free energy surface beyond the TS.     

The mechanism of nucleation for in vitro collagen fibril formation.

The formation of collagen fibrils from soluble monomers and aggregates by thermal gelation at neutral pH can be divided into two distinct stages: a nucleation phase and a growth phase. Turbidity studies of the kinetics of the precipitation reaction show that the lag-phase time or nucleation reaction time, t′_l, is markedly temperature dependent while the growth reaction time is temperature independent. The activation energy of the nucleation reaction is essentially constant over the temperature range studied. In monitoring the nucleation-phase reaction by various physicochemical techniques, including viscosity, sedimentation equilibrium, and light scattering, no evidence for the formation of aggregates was observed. Enrichment of the initial collagen solution with aggregates accelerates nucleation, but de novo nuclei formation is still required even in highly aggregated collagen preparations. Removal of pepsin and pronase susceptible peptides lengthens the nucleation reaction time and increases the sensitivity of the rate of nuclei formation to changes in ionic strength. Electron microscope studies show the fibrils formed from the protease-treated collagen to be less well organized. With pepsin-treated collagen, subfibrils and obliquely striated fibrils are seen, showing that while microfibrils are formed interactions between them are modulated by the enzyme susceptible peptides in the same way that these regions modulate nuclei assembly. It appears that pepsin and pronase susceptible peptide regions of collagen play a more prominent role in the in vitro assembly of collagen molecules to form D-stagger nuclei and fibrils than do ionic interactions between helical molecular regions. A mechanism of nucleation of collagen fibrillogenesis is discussed.     

Identification of the calmodulin binding domain of alpha-fodrin and implications for folding

A cDNA clone producing a protein that binds calmodulin has been isolated from a mouse macrophage library. The cDNA was sequenced and identified as coding for fodrin. By deleting part of the sequence, the calmodulin binding domain was located. The site is situated on repeat 11 of fodrin probably on its extra arm. This part of the sequence exhibits great similarity to other calmodulin binding proteins. Analysis of the sequence and spatial structure of calmodulin revealed a domain which is quite complementary to the sequence identified on fodrin. These results provide a new insight into the structure of fodrin and consequently into the structure of proteins of the spectrin family. A model for the general folding of these molecules is proposed, involving a simple three-layer folding. The structure was further corroborated by analysis of charge distribution in the vicinity of the calmodulin binding site. The folding we propose is in good agreement with digestion experiments and explains observations in diseases resulting from mutations of human spectrin.     

Phosphorylation of the human interleukin-2 receptor and a synthetic peptide identical to its C-terminal, cytoplasmic domain

The recombinant human interleukin-2 (IL-2) receptor was expressed in mouse mammary epithelial cells following the transfection of these cells with an expression vector containing the human IL-2 receptor cDNA. The recombinant IL-2 receptor in these cells was rapidly phosphorylated in response to phorbol myristate acetate (PMA), but its phosphorylation could not be detected in the absence of PMA or upon addition of human IL-2. The C-terminal, cytoplasmic peptide domain of the IL-2 receptor, Gln-Arg-Arg-Gln-Arg-Lys-Ser-Arg-Arg-Thr-Ile, was synthesized and used as a substrate for protein kinase C. The Km for phosphorylation of the peptide by protein kinase C was 23 microM. The stoichiometry of phosphorylation was 1 mol of phosphate/mol of peptide and serine was the predominant amino acid phosphorylated. Because this peptide was a good substrate for protein kinase C in vitro, it was possible that the same serine (serine 247) was also phosphorylated in the receptor in the cell. The IL-2 receptor gene in the expression vector was therefore altered by site-directed mutagenesis to code for an IL-2 receptor containing an alanine in the place of serine 247. The IL-2 receptor expressed by these cells was not phosphorylated in the presence of PMA. These data suggest that protein kinase C, in response to PMA, phosphorylates the C-terminal serine residue (serine 247) in the human IL-2 receptor.     

Solution structure and folding characteristics of the C-terminal SH3 domain of c-Crk-II

Crk-II is a signaling adaptor protein that is involved in many cellular processes including apoptosis, proliferation, and differentiation. It has a modular domain architecture consisting of an Src homology 2 domain (SH2) followed by two Src homology 3 (SH3) domains. The structures and ligand-binding properties of the SH2 and the middle SH3 domains are well-characterized. Several studies suggest that the C-terminal SH3 domain plays an important regulatory role in the protein; however, no structural information is available on this domain, and relatively little is known about its binding partners. In the current work, we have solved the solution NMR structure of the C-terminal SH3 domain. The domain adopts the standard SH3 fold comprising a five-stranded beta barrel. In agreement with alignment and modeling studies, the structure indicates that the canonical-binding surface of the SH3 domain is unusually polar and suggests that this domain may not bind typical PXXP ligands or that it may bind them with reduced affinity. Thermodynamic and kinetic studies show that the domain folds in a reversible two-state manner and that the stability of the fold is similar to that observed for other SH3 domains. These studies offer some insight into the likely structural and thermodynamic consequences of point mutations in the cSH3 domain that are known to deregulate Crk-II function. Our results set the stage for a better understanding the role of the cSH3 domain in the context of the full-length protein.     

DNA and nucleosomes direct distinct folding of a linker histone H1 C-terminal domain

We previously documented condensation of the H1 CTD consistent with adoption of a defined structure upon nucleosome binding using a bulk FRET assay, supporting proposals that the CTD behaves as an intrinsically disordered domain. In the present study, by determining the distances between two different pairs of sites in the C-terminal domain of full length H1 by FRET, we confirm that nucleosome binding directs folding of the disordered H1 C-terminal domain and provide additional distance constraints for the condensed state. In contrast to nucleosomes, FRET observed upon H1 binding to naked DNA fragments includes both intra- and inter-molecular resonance energy transfer. By eliminating inter-molecular transfer, we find that CTD condensation induced upon H1-binding naked DNA is distinct from that induced by nucleosomes. Moreover, analysis of fluorescence quenching indicates that H1 residues at either end of the CTD experience distinct environments when bound to nucleosomes, and suggest that the penultimate residue in the CTD (K195) is juxtaposed between the two linker DNA helices, proposed to form a stem structure in the H1-bound nucleosome.     

Missense mutations define a restricted segment in the C-terminal domain of phytochrome A critical to its regulatory activity.

The phytochrome family of photoreceptors has dual molecular functions: photosensory, involving light signal perception, and regulatory, involving signal transfer to downstream transduction components. To define residues necessary specifically for the regulatory activity of phytochrome A (phyA), we undertook a genetic screen to identify Arabidopsis mutants producing wild-type levels of biologically defective but photochemically active and dimeric phyA molecules. Of eight such mutants identified, six contain missense mutations (including three in the same residue, glycine 727) clustered within a restricted segment in the C-terminal domain of the polypeptide. Quantitative photobiological analysis revealed retention of varying degrees of partial activity among the different alleles--a result consistent with the extent of conservation at the position mutated. Together with additional data, these results indicate that the photoreceptor subdomain identified here is critical to the regulatory activity of both phyA and phyB.     

Effects of mutations on the thermodynamics of a protein folding reaction: implications for the mechanism of formation of the intermediate and transition states

We have measured changes in heat capacity, entropy, and enthalpy for each step in the folding reaction of CD2.d1 and evaluated the effects of core mutations on these properties. All wild-type and mutant forms fold through a rapidly formed intermediate state that precedes the rate-limiting transition state. Mutations have a pronounced effect on the enthalpy of both the intermediate and folded states, but in all cases a compensatory change in entropy results in a small net free-energy change. While the enthalpy change in the folded state can be attributed to a loss of van der Waals interactions, it has already been shown that changes in the stability of the intermediate are dominated by changes in secondary structure propensity [Lorch et al. (1999) Biochemistry 38, 1377-1385]. It follows that the thermodynamic basis of beta-propensity is enthalpic in origin. The effects of mutations on the enthalpy and entropy of the transition state are smaller than on the ground states. This relative insensitivity to mutation is discussed in the light of theories concerning the nature of the rate-limiting barrier in folding reactions.     

Detection of a hidden folding intermediate in the focal adhesion target domain: Implications for its function and folding

The focal adhesion target (FAT) domain of focal adhesion kinase has a four-helix bundle structure. Based on a hydrogen exchange-constrained computer simulation study and some indirect experimental results, it has been suggested that a partially unfolded state of the FAT domain with the N-terminal helix unfolded plays an important role in its biological function. Here, using a native-state hydrogen exchange method, we directly detected an intermediate with the N-terminal helix unfolded in a mutant (Y925E) of the FAT domain. In addition, kinetic folding studies on the FAT domain suggest that this intermediate exists on the native side of the rate-limiting transition state for folding. These results provide more direct evidence of the existence of the proposed intermediate and help to understand the folding mechanism of small single domain proteins.     

Crystal structure of the measles virus phosphoprotein domain responsible for the induced folding of the C-terminal domain of the nucleoprotein

Measles virus is a negative-sense, single-stranded RNA virus belonging to the Mononegavirales order which comprises several human pathogens such as Ebola, Nipah, and Hendra viruses. The phosphoprotein of measles virus is a modular protein consisting of an intrinsically disordered N-terminal domain (Karlin, D., Longhi, S., Receveur, V., and Canard, B. (2002) Virology 296, 251-262) and of a C-terminal moiety (PCT) composed of alternating disordered and globular regions. We report the crystal structure of the extreme C-terminal domain (XD) of measles virus phosphoprotein (aa 459-507) at 1.8 A resolution. We have previously reported that the C-terminal domain of measles virus nucleoprotein, NTAIL, is intrinsically unstructured and undergoes induced folding in the presence of PCT (Longhi, S., Receveur-Brechot, V., Karlin, D., Johansson, K., Darbon, H., Bhella, D., Yeo, R., Finet, S., and Canard, B. (2003) J. Biol. Chem. 278, 18638-18648). Using far-UV circular dichroism, we show that within PCT, XD is the region responsible for the induced folding of NTAIL. The crystal structure of XD consists of three helices, arranged in an anti-parallel triple-helix bundle. The surface of XD formed between helices alpha2 and alpha3 displays a long hydrophobic cleft that might provide a complementary hydrophobic surface to embed and promote folding of the predicted alpha-helix of NTAIL. We present a tentative model of the interaction between XD and NTAIL. These results, beyond presenting the first measles virus protein structure, shed light both on the function of the phosphoprotein at the molecular level and on the process of induced folding.     

The thermal transition of a non-hydroxylated form of collagen. Evidence for a role for hydroxyproline in stabilizing the triple-helix of collagen

Protocollagen, a non-hydroxylated form of collagen, was extracted with cold 0.1 N acetic acid from embryonic tendon cells incubated with α,α′-dipyridyl and the protein was purified by controlled proteolytic digestion. The resulting modified protocollagen was shown to consist of polypeptides the same size as α1 and α2 chains of collagen and had a thermal transition by optical rotation similar to collagen. The T_m however was 24°, a value which was 15° lower than the T_m of an hydroxylated form of collagen from the same source. The results suggest that hydroxylated proline increases the thermal stability of collagen.