GANDivAWeb: A web server for detecting early folding units ("foldons") from protein 3D structures

Background  

It has long been known that small regions of proteins tend to fold independently and are then stabilized by interactions between these distinct subunits or modules. Such units, also known as autonomous folding units (AFUs) or"foldons" play a key role in protein folding. A knowledge of such early folding units has diverse applications in protein engineering as well as in developing an understanding of the protein folding process. Such AFUs can also be used as model systems in order to study the structural organization of proteins.     

Probing minimal independent folding units in dihydrofolate reductase by molecular dissection.

Molecular dissection was employed to identify minimal independent folding units in dihydrofolate reductase (DHFR) from Escherichia coli. Eight overlapping fragments of DHFR, spanning the entire sequence and ranging in size from 36 to 123 amino acids, were constructed by chemical cleavage. These fragments were designed to examine the effect of tethering multiple elements of secondary structure on folding and to test if the secondary structural domains represent autonomous folding units. CD and fluorescence spectroscopy demonstrated that six fragments containing up to a total of seven alpha-helices or beta-strands and, in three cases, the adenine binding domain (residues 37-86), are largely disordered. A stoichiometric mixture of the two fragments comprising the large discontinuous domain, 1-36 and 87-159, also showed no evidence for folding beyond that observed for the isolated fragments. A fragment containing residues 1-107 appears to have secondary and tertiary structure; however, spontaneous self-association made it impossible to determine if this structure solely reflects the behavior of the monomeric form. In contrast, a monomeric fragment spanning residues 37-159 possesses significant secondary and tertiary structure. The urea-induced unfolding of fragment 37-159 in the presence of 0.5 M ammonium sulfate was found to be a well-defined, two-state process. The observation that fragment 37-159 can adopt a stable native fold with unique, aromatic side-chain packing is quite striking because residues 1-36 form an integral part of the structural core of the full-length protein.     

Generation of a consensus protein domain dictionary

MOTIVATION: The discovery of new protein folds is a relatively rare occurrence even as the rate of protein structure determination increases. This rarity reinforces the concept of folds as reusable units of structure and function shared by diverse proteins. If the folding mechanism of proteins is largely determined by their topology, then the folding pathways of members of existing folds could encompass the full set used by globular protein domains. RESULTS: We have used recent versions of three common protein domain dictionaries (SCOP, CATH and Dali) to generate a consensus domain dictionary (CDD). Surprisingly, 40% of the metafolds in the CDD are not composed of autonomous structural domains, i.e. they are not plausible independent folding units. This finding has serious ramifications for bioinformatics studies mining these domain dictionaries for globular protein properties. However, our main purpose in deriving this CDD was to generate an updated CDD to choose targets for MD simulation as part of our dynameomics effort, which aims to simulate the native and unfolding pathways of representatives of all globular protein consensus folds (metafolds). Consequently, we also compiled a list of representative protein targets of each metafold in the CDD. Availability and implementation: This domain dictionary is available at www.dynameomics.org.     

Reconstitution of a native-like SH2 domain from disordered peptide fragments examined by multidimensional heteronuclear NMR

The N-terminal SH2 domain from the p85alpha subunit of phosphatidylinositol 3' kinase is cleaved specifically into 9- and 5-kD fragments by limited proteolytic digestion with trypsin. The noncovalent SH2 domain complex and its constituent tryptic peptides have been investigated using high-resolution heteronuclear magnetic resonance (NMR). These studies have established the viability of the SH2 domain as a fragment complementation system. The individual peptide fragments are predominantly unstructured in solution. In contrast, the noncovalent 9-kD + 5-kD complex shows a native-like (1)H-(15)N HSQC spectrum, demonstrating that the two fragments fold into a native-like structure on binding. Chemical shift analysis of the noncovalent complex compared to the native SH2 domain reveals that the highest degree of perturbation in the structure occurs at the cleavage site within a flexible loop and along the hydrophobic interface between the two peptide fragments. Mapping of these chemical shift changes on the structure of the domain reveals changes consistent with the reduction in affinity for the target peptide ligand observed in the noncovalent complex relative to the intact protein. The 5-kD fragment of the homologous Src protein is incapable of structurally complementing the p85 9-kD fragment, either in complex formation or in the context of the full-length protein. These high-resolution structural studies of the SH2 domain fragment complementation features establish the suitability of the system for further protein-folding and design studies.     

The domain organization of streptokinase: nuclear magnetic resonance, circular dichroism, and functional characterization of proteolytic fragments.

Streptococcus equisimilis streptokinase (SK) is a bacterial protein of unknown tertiary structure and domain organization that is used extensively to treat acute myocardial infarction following coronary thrombosis. Six fragments of SK were generated by limited proteolysis with chymotrypsin and purified. NMR and CD experiments have shown that the secondary and tertiary structure present in the native molecule is preserved within all fragments, except the N-terminal fragment SK7. NMR spectra demonstrate the presence in SK of three structurally autonomous domains and a less structured C-terminal "tail." Cleavage within the N-terminal domain generates an N-terminal fragment, SK7, which remains noncovalently associated with the remainder of the molecule; in isolation, SK7 adopts an unfolded conformation. The abilities of these fragments to induce active site formation within human plasminogen upon formation of their heterodimeric complex were assayed. The lowest mass SK fragment exhibiting Plg-dependent activator activity was shown to be SK27 (mass 27,000, residues 147-380), which contains both central and C-terminal domains, although this activity was reduced approximately 6,000-fold relative to that of full-length SK. The activity of a 36,000 mass fragment, SK36 (residues 64-380), which differs from SK27 in possessing a portion of the N-terminal domain, was reduced to 0.1-1.0% of that of SK. Other fragments (masses 7,000, 11,000, 16,000, 17,000, 25,000, and 26,000), representing either single domains or single domains extended by portions of other domains, were inactive. However, SK7 (residues 1-63), at a 100-fold molar excess concentration, greatly potentiated the activities of SK27 and SK36, by up to 50- and > 130-fold, respectively. These findings demonstrate that all of SK's three domains are essential for native-like SK activity. The central and C-terminal domains mediate plasminogen-binding and active site-generating functions, whereas the N-terminal domain mediates an activity-potentiating function.     

Molecular dissection of the folding mechanism of the alpha subunit of tryptophan synthase: an amino-terminal autonomous folding unit controls several rate-limiting steps in the folding of a single domain protein.

The alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli is a 268-residue 8-stranded beta/alpha barrel protein. Two autonomous folding units, comprising the first six strands (residues 1-188) and the last two strands (residues 189-268), have been previously identified in this single structural domain protein by tryptic digestion [Higgins, W., Fairwell, T., and Miles, E. W. (1979) Biochemistry 18, 4827-4835]. The larger, amino-terminal fragment, alphaTS(1-188), was overexpressed and independently purified, and its equilibrium and kinetic folding properties were studied by absorbance, fluorescence, and near- and far-UV circular dichroism spectroscopies. The native state of the fragment unfolds cooperatively in an apparent two-state transition with a stability of 3.98 +/- 0.19 kcal mol(-1) in the absence of denaturant and a corresponding m value of 1.07 +/- 0.05 kcal mol(-1) M(-1). Similar to the full-length protein, the unfolding of the fragment shows two kinetic phases which arise from the presence of two discrete native state populations. Additionally, the fragment exhibits a significant burst phase in unfolding, indicating that a fraction of the folded state ensemble under native conditions has properties similar to those of the equilibrium intermediate populated at 3 M urea in full-length alphaTS. Refolding of alphaTS(1-188) is also complex, exhibiting two detectable kinetic phases and a burst phase that is complete within 5 ms. The two slowest isomerization phases observed in the refolding of the full-length protein are absent in the fragment, suggesting that these phases reflect contributions from the carboxy-terminal segment. The folding mechanism of alphaTS(1-188) appears to be a simplified version of the mechanism for the full-length protein [Bilsel, O., Zitzewitz, J. A., Bowers, K.E, and Matthews, C. R.(1999) Biochemistry 38, 1018-1029]. Four parallel channels in the full-length protein are reduced to a pair of channels that most likely reflect a cis/trans proline isomerization reaction in the amino-terminal fragment. The off- and on-pathway intermediates that exist for both full-length alphaTS and alphaTS(1-188) may reflect the preponderance of local interactions in the beta/alpha barrel motif.     

Identifying protein domains by global analysis of soluble fragment data

The production and analysis of individual structural domains is a common strategy for studying large or complex proteins, which may be experimentally intractable in their full-length form. However, identifying domain boundaries is challenging if there is little structural information concerning the protein target. One experimental procedure for mapping domains is to screen a library of random protein fragments for solubility, since truncation of a domain will typically expose hydrophobic groups, leading to poor fragment solubility. We have coupled fragment solubility screening with global data analysis to develop an effective method for identifying structural domains within a protein. A gene fragment library is generated using mechanical shearing, or by uracil doping of the gene and a uracil-specific enzymatic digest. A split green fluorescent protein (GFP) assay is used to screen the corresponding protein fragments for solubility when expressed in Escherichia coli. The soluble fragment data are then analyzed using two complementary approaches. Fragmentation “hotspots” indicate possible interdomain regions. Clustering algorithms are used to group related fragments, and concomitantly predict domain location. The effectiveness of this Domain Seeking procedure is demonstrated by application to the well-characterized human protein p85α.     

Heme as an optical probe of a conformational transition of ovine recPrP

The prion protein occurs as a globular domain and a leading fragment whose structure is not well-defined. For the ovine species, all of the tryptophan residues are in the initial fragment, while the globular domain is rich in tyrosine residues. Using heme as a spectroscopic probe, we have studied the recombinant prion protein before and after a temperature-induced conformational change. As for most heme proteins, the absorption spectrum of heme-CO displays a red shift upon binding to the protein, and both the Y and W fluorescence are highly quenched. Flash photolysis kinetics of the PrP-heme-CO complex shows a low yield for the bimolecular phase, indicating a pocket around the hemes. By comparing the holoprotein and the truncated sequence corresponding to the globular domain, the stoichiometry was determined to be five hemes for the globular domain and two hemes for the leading fragment. At high temperature, the hemes are released; upon cooling, only two hemes bind, and only the tryptophan fluorescence is quenched; this would indicate that the globular domain has formed a more compact structure, which is inert with respect to the hydrophobic probe. The final state of polymerization is perturbed if the synthetic peptide "N3" (PrP residues 142-166, which include the first helix) is added to the prion protein solution; the temperature cycle no longer reduces the number of heme binding sites. This would indicate that the peptide may alter or inhibit the polymer formation.     

Identification, expression, and purification of a unique stable domain from human HSPC144 protein

HSPC144 is a newly identified gene in human CD34(+) hematopoietic stem/progenitor cells. In this work, we have expressed and purified the 225-residue protein from Escherichia coli BL21 (DE3) and identified a stable fragment HSPC144-P (residues 44-225) by limited proteolysis method. The HSPC144-P fragment exhibits high stability with a little increase of secondary structure percentage as compared with the full-length protein. We anticipated that the N-terminally truncated protein possesses a more compact structure. By sequence analysis, the proteolytic fragment shares a great similarity with DUF589 domain, a previously identified domain with unknown function. This novel domain is highly conserved in Thy28 proteins and is worthy of structural and functional studies. We have subcloned this homologous domain from HSPC144 protein and purified to homogeneity for structure analysis. The (15)N and (15)N/(13)C-labeled DUF589 domain samples have been prepared successfully and determination of the NMR structure is in progress.     

Domain characteristics of the carboxyl-terminal fragment 206-316 of thermolysin

The existence, location, and characteristics of protein domains have been investigated by studying the structural properties of the carboxyl-terminal cyanogen bromide fragment 206–316 of thermolysin. As judged by far-uv CD measurements in aqueous solution under neutral conditions, the fragment attains a substantial degree of α-helical structure comparable to that exhibited by the corresponding region in native thermolysin. By radioimmunoassay techniques, a considerable degree of nativeness of fragment conformation has been deduced from comparison of the relative affinities of thermolysin and fragment 206–316 for antibodies specific for the 206–316 region in the intact protein. The fragment shows noteworthy stability to protein denaturants. The overall spectroscopic and immunochemical data suggest that fragment 206–316 is able to refold into a stable, nativelike structure independently from the rest of the molecule, thus providing support for the view that this fragment may contain a substantial part, if not all, of a protein domain structure.     

Independent folding of individual components in hybrid proteins. Evidence that the carboxy-terminal 135 residues of the LexA repressor constitute a single autonomous domain

Inactivation of the Escherichia coli repressor protein, LexA, takes place through a cleavage reaction which hydrolyzes the Ala84-Gly85 peptide bond near the center of the molecule. The mechanism of cleavage has previously been shown to be an intramolecular reaction stimulated in vitro by elevated pH or by the addition of activated RecA protein. The entire self-cleavage activity of LexA has been found to lie within a 135-residue tryptic fragment extending from Leu68 to the end of the protein at Leu202. Since the activity of self-cleavage is dependent on the proper three-dimensional structure of the protein, we have used it as a probe to investigate the extend of folding autonomy and functional independence of this 135-residue carboxy-terminal domain of LexA by applying a protein fusion approach. A series of twelve different hybrid proteins, containing LexA sequences in a variety of predefined primary structural arrangements, were constructed and evaluated for whether or not self-cleavage activity has been retained. The results revealed that retention or loss of activity is independent of the nature or size of the foreign protein used. Loss of self-cleavage was found to be a function of amino- or carboxy-terminal deletions in the self-cleaving LexA component of the fusion proteins. The present findings, together with the observations of other artificial fusions proteins and the naturally occurring bifunctional and multifunctional proteins, along with the data on helix packing, provide further support for the notion of modular architecture of proteins and suggest that when these autonomous units are fused, they retain their tendency to fold independently of the remainder of the polypeptide to generate physically linked active domains, rather than to fold dependently and yield scrambled structures.     

Crystal structure of a dimerization domain of human Caprin-2: similar overall dimeric fold but different molecular surface properties to that of human Caprin-1

Abstract

Human Caprin-1 and Caprin-2 are prototypic members of the caprin (cytoplasmic activation/proliferation-associated protein) protein family. Vertebrate caprin proteins contain two highly conserved homologous regions (HR1 and HR2) and C-terminal RGG motifs. Drosophila caprin (dCaprin) shares HR1 and RGG motifs but lacks HR2. Caprin-1 and Caprin-2 have important and non-redundant functions. The detailed molecular mechanisms of their actions remain largely unknown. Previously, we determined the crystal structure of a ～120-residue fragment of Caprin-1 within the HR1 region. The structure has a novel all α-helical fold that self-associates to form a homodimer. In this study, the crystal structure of a corresponding fragment from Caprin-2 is reported. The Caprin-2 fragment has similar protein fold and dimeric structure as that of the Caprin-1 fragment. Structural comparison reveals that the molecular interactions mediating homodimerization of Caprin-1 and Caprin-2 are largely conserved in the two systems. Structural-modelling study of the corresponding dCaprin fragment indicates that dCaprin may also adopt a similar dimeric structure. The presence of a dimerization domain within HR1 may represent an evolutionarily conserved feature of the caprin protein family. Interestingly, while Caprin-1 and Caprin-2 adopt similar overall dimeric structures, the two structures have quite different molecular surface properties. In the Caprin-1 dimeric structure, some of the surface areas are known or suspected to function as binding sites for Carpin-1-interacting proteins. The different surface properties of the caprin dimeric structures may dictate their intermolecular interaction with specific protein partners.

Communicated by Ramaswamy H. Sarma.     

Peptide fragment studies on the folding elements of dihydrofolate reductase from Escherichia coli

One of the necessary conditions for a protein to be foldable is the presence of a complete set of “folding elements” (FEs) that are short, contiguous peptide segments distributed over an amino acid sequence. The FE‐assembly model of protein folding has been proposed, in which the FEs play a role in guiding structure formation through FE–FE interactions early in folding. However, two major issues remain to be clarified regarding the roles of the FEs in determining protein foldability. Are the FEs AFUs that can form nativelike structures in isolation? Is the presence of only the FEs without mutual connections a sufficient condition for a protein to be foldable? Here, we address these questions using peptide fragments corresponding to the FEs of DHFR from Escherichia coli. We show by CD measurement that the FE peptides are unfolded under the native conditions, and some of them have the propensities toward non‐native helices. MD simulations also show the non‐native helical propensities of the peptides, and the helix contents estimated from the simulations are well correlated with those estimated from the CD in TFE. Thus, the FEs of DHFR are not AFUs, suggesting the importance of the FEs in nonlocal interactions. We also show that equimolar mixtures of the FE peptides do not induce any structural formation. Therefore, mutual connections between the FEs, which should strengthen the nonlocal FE–FE interactions, are also one of the necessary conditions for a protein to be foldable. Proteins 2006. © 2005 Wiley‐Liss, Inc.     

A recombinant glycine receptor fragment forms homo-oligomers distinct from its GABA(A) counterpart

The ligand-gated ion channel receptor superfamily includes receptors for glycine, GABA, acetylcholine and serotonin. Whereas the acetylcholine and serotonin receptors mediate excitory neurotransmissions, both glycine and GABA(A) receptors are inhibitory. In this study, a fragment of the human glycine receptor alpha1 subunit, consisting of residues Ala165-Met291 (numbering based on the precursor protein), was hyperexpressed for the first time in Escherichia coli. This fragment is highly homologous in sequence to the corresponding fragment of the GABA(A) receptor. The recombinant fragment was found to have stable beta-rich secondary structure, similar to that found for the homologous GABA(A) receptor fragment, and ordered tertiary packing, suggesting a stable structural domain. Results from laser scattering studies suggest that the fragment forms trimers in solution. In addition, SDS-induced changes in secondary structure were found to occur prior to changes in oligomerization status, suggesting that oligomerization was secondary structure dependent. A study of quaternary structure using single particle analysis electron microscopy (EM) also suggested that the fragment formed homo-trimers. One trimer measures approximately 7.5 nm in diameter with a central cavity approximately 1.5 nm across. This is the first EM study on a single domain of the glycine receptor and the result is in contrast to the pentameric assembly of the equivalent GABA(A) receptor fragment reported by us earlier. The fact that this fragment alone could form oligomers in vitro suggests that amino acid residues within this segment may be involved in the oligomerization of the glycine receptor in vivo. Furthermore, the finding that two cousin receptor fragments form distinct quaternary structures indicates that sequence similarity does not necessarily imply quaternary structure similarity and, hence, care must be taken when applying a structure model derived from studies of individual receptors to the whole ligand-gated ion channel superfamily.     

Autonomous folding of the excised coenzyme-binding domain of D-glyceraldehyde 3-phosphate dehydrogenase from Thermotoga maritima.

An important question in protein folding is whether compact substructures or domains are autonomous units of folding and assembly. The protomer of the tetrameric D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima has a complex coenzyme-binding domain, in which residues 1-146 form a compact substructure with the last 31 residues (313-333). Here it is shown that the gene of a single-chain protein can be expressed in Escherichia coli after deleting the 163 codons corresponding to the interspersed catalytic domain (150-312). The purified gene product is a soluble, monomeric protein that binds both NAD+ and NADH strongly and possesses the same unfolding transition induced by guanidinium chloride as the native tetramer. The autonomous folding of the coenzyme-binding domain has interesting implications for the folding, assembly, function, and evolution of the native enzyme.     

Crystal Structure of P58(IPK) TPR Fragment Reveals the Mechanism for its Molecular Chaperone Activity in UPR

P58(IPK) might function as an endoplasmic reticulum molecular chaperone to maintain protein folding homeostasis during unfolded protein responses. P58(IPK) contains nine tetratricopeptide repeat (TPR) motifs and a C-terminal J-domain within its primary sequence. To investigate the mechanism by which P58(IPK) functions to promote protein folding within the endoplasmic reticulum, we have determined the crystal structure of P58(IPK) TPR fragment to 2.5 Å resolution by the SAD method. The crystal structure of P58(IPK) revealed three domains (I-III) with similar folds and each domain contains three TPR motifs. An ELISA assay indicated that P58(IPK) acts as a molecular chaperone by interacting with misfolded proteins such as luciferase and rhodanese. The P58(IPK) structure reveals a conserved hydrophobic patch located in domain I that might be involved in binding the misfolded polypeptides. Structure-based mutagenesis for the conserved hydrophobic residues located in domain I significantly reduced the molecular chaperone activity of P58(IPK).     

Hierarchic organization of globular proteins: Experimental studies on thermolysin

Previous studies from this laboratory have shown that the thermolysin fragment 121–316, comprising entirely the“all-α” COOH-terminal structural domain 158–316, as well as fragment 206–316 (fragment FII) are able to refold into a native-like, stable structure independently from the rest of the protein molecule. The present report describes conformational properties of fragments 228–316 and 255–316 obtained by chemical and enzymatic cleavage of fragment FII, respectively. These subfragments are able to acquire a stable conformation of native-like characteristics, as judged by quantitative analysis of secondary structure from far-ultra-violet circular dichroism spectra and immunochemical properties using rabbit anti-thermolysin antibodies. Melting curves of the secondary structure of the fragments show cooperativity with a temperature of half-denaturationT _mof 65–66°C. The results of this study provide evidence that it is possible to isolate stable supersecondary structures (folding units) of globular proteins and correlate well with predictions of subdomains of the COOH-terminal structural domain 158–316 of thermolysin.     

When a domain is not a domain,and why it is important to properly filter proteins in databases

Membership in a protein domain database does not a domain make; a feature we realized when generating a consensus view of protein fold space with our consensus domain dictionary (CDD). This dictionary was used to select representative structures for characterization of the protein dynameome: the Dynameomics initiative. Through this endeavor we rejected a surprising 40% of the 1,695 folds in the CDD as being non‐autonomous folding units. Although some of this was due to the challenges of grouping similar fold topologies, the dissonance between the cataloguing and structural qualification of protein domains remains surprising. Another potential factor is previously overlooked intrinsic disorder; predictions suggest that 40% of proteins have either local or global disorder. One thing is clear, filtering a structural database and ensuring a consistent definition for protein domains is crucial, and caution is prescribed when generalizations of globular domains are drawn from unfiltered protein domain datasets.     

Tec家族蛋白酪氨酸激酶Itk PH结构域与OS-9蛋白的相互作用

用酵母双杂交技术筛选与ItkPH结构域相互作用的蛋白分子 ,以了解Itk的功能及其在T细胞信号转导中的位置与作用 .Itk的PH结构域扩增后克隆入酵母双杂交系统的pLexA载体 ,转化酵母细胞EGY4 8(p8op lacZ) ,经检测PH结构域无自激活作用 ,且对酵母细胞无毒性作用 .用PH结构域作为“钓饵”蛋白 ,在酵母双杂交系统中筛选构建于AD载体的T细胞cDNA文库 .将PH结构域及筛库所得基因片段分别进行融合表达 ,用于体外结合实验 ,进一步证实二者的相互作用 .经营养缺陷选择、诱导筛选和鉴定确证 ,筛库所得的插段约 15 0 0bp的文库质粒为一真阳性克隆 .经blast比较分析为骨肉瘤、横纹肌肉瘤等肿瘤组织中高表达的os 9基因 .体外结合实验也表明 ,ItkPH结构域可与该基因表达产物结合 .Itk的PH结构域可与OS 9蛋白相互作用 .二者结合的意义有待进一步研究