Statistical analysis of unstructured amino acid residues in protein structures

We have performed a statistical analysis of unstructured amino acid residues in protein structures available in the databank of protein structures. Data on the occurrence of disordered regions at the ends and in the middle part of protein chains have been obtained: in the regions near the ends (at distance less than 30 residues from the N- or C-terminus), there are 66% of unstructured residues (38% are near the N-terminus and 28% are near the C-terminus), although these terminal regions include only 23% of the amino acid residues. The frequencies of occurrence of unstructured residues have been calculated for each of 20 types in different positions in the protein chain. It has been shown that relative frequencies of occurrence of unstructured residues of 20 types at the termini of protein chains differ from the ones in the middle part of the protein chain; amino acid residues of the same type have different probabilities to be unstructured in the terminal regions and in the middle part of the protein chain. The obtained frequencies of occurrence of unstructured residues in the middle part of the protein chain have been used as a scale for predicting disordered regions from amino acid sequence using the method (FoldUnfold) previously developed by us. This scale of frequencies of occurrence of unstructured residues correlates with the contact scale (previously developed by us and used for the same purpose) at a level of 95%. Testing the new scale on a database of 427 unstructured proteins and 559 completely structured proteins has shown that this scale can be successfully used for the prediction of disordered regions in protein chains.     

Selection of antibody fragments specific for an alpha-helix region of acylphosphatase

The native state of common-type acylphosphatase (AcP) elicits two alpha-helices spanning residues 22-32 and 55-67 in the protein sequence. A peptide corresponding to the second alpha-helix (helix-2) of the protein was used to select phage antibodies consisting of a single chain fragment variable. The selection was performed in the presence of trifluoroethanol, a cosolvent known to induce the formation of helical structure in peptides and proteins. Phage scFv antibodies capable of binding the peptide specifically in a trifluoroethanol-induced alpha-helical conformation were isolated by affinity selection (biopanning). Some of these scFvs were also able to bind the native protein but not the peptide in a non-helical unstructured state. This indicates that the structural determinant recognized by the selected antibodies is the alpha-helical conformation of this specific region, rather than simply its amino acid sequence. This study shows that phage display libraries can be used to raise antibodies one can use as reagents able to target regions of a protein with a specific native-like secondary structure.     

Kinetic stability and crystal structure of the viral capsid protein SHP

SHP, the capsid-stabilizing protein of lambdoid phage 21, is highly resistant against denaturant-induced unfolding. We demonstrate that this high functional stability of SHP is due to a high kinetic stability with a half-life for unfolding of 25 days at zero denaturant, while the thermodynamic stability is not unusually high. Unfolding experiments demonstrated that the trimeric state (also observed in crystals and present on the phage capsid) of SHP is kinetically stable in solution, while the monomer intermediate unfolds very rapidly. We also determined the crystal structure of trimeric SHP at 1.5A resolution, which was compared to that of its functional homolog gpD. This explains how a tight network of H-bonds rigidifies crucial interpenetrating residues, leading to the observed extremely slow trimer dissociation or denaturation. Taken as a whole, our results provide molecular-level insights into natural strategies to achieve kinetic stability by taking advantage of protein oligomerization. Kinetic stability may be especially needed in phage capsids to allow survival in harsh environments.     

p53 contains large unstructured regions in its native state

The human tumor suppressor protein p53 is understood only to some extent on a structural level. We performed a comprehensive biochemical and biophysical structure-function analysis of p53 full-length protein and p53 fragments. The analysis showed that p53 and the fragments investigated form stable functional units. Full-length p53 and the tetrameric fragment N93p53 (residues 93-393) are, however, destabilized significantly compared to the monomeric core domain (residues 94-312) and the monomeric fragment p53C312 (residues 1-312). At the physiological temperature of 37 degrees C and in the absence of modifications or stabilizing partners, wild-type p53 is more than 50% unfolded correlating with a 75% loss in DNA-binding activity. Furthermore the analysis of CD spectra revealed that full-length p53 contains large unstructured regions in its N and C-terminal parts. Our results indicate that full-length p53 is a modular protein consisting of defined structured and unstructured regions. We propose that p53 belongs to the growing family of loosely folded or partially unstructured native proteins. The lack of a rigid structure combined with the low overall stability may allow the physiological interaction of p53 with a multitude of partner proteins and the regulation of its turnover.     

Comprehensive mutagenesis of the C-terminal domain of the M13 gene-3 minor coat protein: the requirements for assembly into the bacteriophage particle

Filamentous bacteriophage assemble at the host membrane in a non-lytic process; the gene-3 minor coat protein (P3) is required for release from the membrane and subsequently, for recognition and infection of a new host. P3 contains at least three distinct domains: two N-terminal domains that mediate host recognition and infection, and a C-terminal domain (P3-C) that is required for release from the host cell following phage assembly and contributes to the structural stability of the phage particle. A comprehensive mutational analysis of the 150 residue P3-C revealed that only 24 side-chains, located within the last 70 residues of sequence, were necessary for efficient incorporation into a wild-type coat. The results reveal that the requirements for the assembly of P3 into the phage particle are quite lax and involve only a few key side-chains. These findings shed light on the functional and structural requirements for filamentous phage assembly, and they may provide guidelines for the engineering of improved coat proteins as scaffolds for phage display technology.     

Functional Role of the N-Terminal Domain of Bacteriophage T4-Gene Product 11

Bacteriophage T4 late gene product 11 (gp11), the three-dimensional structure of which has been solved by us to 2.0 A resolution, is a part of the virus' baseplate. The gp11 polypeptide chain consists of 219 amino acid residues and the functionally active protein is a three-domain homotrimer. In this work, we have studied the role of gp11 N-terminal domain in the formation of a functionally active trimer. Deletion variants of gp11 and monoclonal antibodies recognizing the native conformation of gp11 trimer have been selected. Long deletions up to a complete removal of the N-terminal domain, containing 64 residues, do not affect the gp11 trimerization, but considerably change the protein structure and lead to the loss of its ability to incorporate into the baseplate. However, the deletion of the first 17 N-terminal residues results in functionally active protein that can complete the 11(-)-defective phage particles in in vitro complementation assay. This region of the polypeptide chain is probably essential for gp11-gp10 stable complex formation at the early stages of phage baseplate assembly in vivo. A study of the gp10 deletion variants suggests that the central domain of gp10 trimer is responsible for the interaction with gp11.     

Specific residues in plasmatocyte-spreading peptide are required for receptor binding and functional antagonism of insect immune cells

Plasmatocyte-spreading peptide (PSP) is a 23-amino acid cytokine that activates a class of insect immune cells called plasmatocytes. PSP consists of two regions: an unstructured N terminus (1-6) and a highly structured core (7-23). Prior studies identified specific residues in both the structured and unstructured regions required for biological activity. Most important for function were Arg13, Phe3, Cys7, Cys19, and the N-terminal amine of Glu1. Here we have built on these results by conducting cell binding and functional antagonism studies. Alanine replacement of Met12 (M12A) resulted in a peptide with biological activity indistinguishable from PSP. Competitive binding experiments using unlabeled and 125I-M12A generated an IC50 of 0.71 nm and indicated that unlabeled M12A, at concentrations > or =100 nm, completely blocked binding of label to hemocytes. We then tested the ability of other peptide mutants to displace 125I-M12A at a concentration of 100 nm. In the structured core, we found that Cys7 and Cys19 were essential for cell binding and functional antagonism, but these effects were likely because of the importance of these residues for maintaining the tertiary structure of PSP. Arg13, in contrast, was also essential for binding and activity but is not required for maintenance of structure. In the unstructured N-terminal region, deletion of the phenyl group from Phe3 yielded a peptide that reduced binding of 125I-M12A 326-fold. This and all other mutants of Phe3 we bioassayed were unable to antagonize PSP. Deletion of Glu1 in contrast had almost no effect on binding and was a strong functional antagonist. Experiments using a photoaffinity analog indicated that PSP binds to a single 190-kDa protein.     

A novel approach to segregate and identify functional loop regions in protein structures using their Ramachandran maps

The loops which connect or flank helices/sheets in protein structures are known to be functionally important. However, ironically they also belong to the part of protein whose structure is least accurately predicted. Here, a new method to isolate and analyze loop regions in protein structure is proposed using the spatial coordinates of the solved three‐dimensional structure. The extent of dispersion among points of successive amino acid residues in the Ramachandran map of protein region is utilized to calculate the Mean Separation between these points in the Ramachandran Plot (MSRP). Based on analysis of 2935 protein secondary structure regions obtained using DSSP software, spanning a range from 2 to 64 residues, taken from a set of 170 proteins, it is shown that helices (MSRP < 17) and strands (MSRP < 64) stand effectively demarcated from the loop regions (MSRP > 130). Analysis of 43 DNA binding and 98 ligand binding proteins revealed several loop regions with clear change in MSRP subsequent to binding. The population of such loops correlated with the magnitude of backbone displacement in the protein subsequent to binding. Can changes in MSRP quantify the temporal oscillations in dihedral angles among structured/unstructured regions in proteins? Molecular dynamics simulations (10 ns) revealed that deviations in MSRP among different snapshots in the trajectory were at least twofold higher for unstructured proteins in comparison with ordered proteins. The above results validate the use of MSRP parameter as a tool to identify and investigate functionally active loops and unstructured regions in protein structures. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Structure of apolipoprotein A-I N terminus on nascent high density lipoproteins

Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins (HDL) and a critical element of cholesterol metabolism. To better elucidate the role of the apoA-I structure-function in cholesterol metabolism, the conformation of the apoA-I N terminus (residues 6-98) on nascent HDL was examined by electron paramagnetic resonance (EPR) spectroscopic analysis. A series of 93 apoA-I variants bearing single nitroxide spin label at positions 6-98 was reconstituted onto 9.6-nm HDL particles (rHDL). These particles were subjected to EPR spectral analysis, measuring regional flexibility and side chain solvent accessibility. Secondary structure was elucidated from side-chain mobility and molecular accessibility, wherein two major α-helical domains were localized to residues 6-34 and 50-98. We identified an unstructured segment (residues 35-39) and a β-strand (residues 40-49) between the two helices. Residues 14, 19, 34, 37, 41, and 58 were examined by EPR on 7.8, 8.4, and 9.6 nm rHDL to assess the effect of particle size on the N-terminal structure. Residues 14, 19, and 58 showed no significant rHDL size-dependent spectral or accessibility differences, whereas residues 34, 37, and 41 displayed moderate spectral changes along with substantial rHDL size-dependent differences in molecular accessibility. We have elucidated the secondary structure of the N-terminal domain of apoA-I on 9.6 nm rHDL (residues 6-98) and identified residues in this region that are affected by particle size. We conclude that the inter-helical segment (residues 35-49) plays a role in the adaptation of apoA-I to the particle size of HDL.     

Structure of the coat protein in Pf1 bacteriophage determined by solid-state NMR spectroscopy

The atomic resolution structure of Pf1 coat protein determined by solid-state NMR spectroscopy of magnetically aligned filamentous bacteriophage particles in solution is compared to the structures previously determined by X-ray fiber and neutron diffraction, the structure of its membrane-bound form, and the structure of fd coat protein. These structural comparisons provide insights into several biological properties, differences between class I and class II filamentous bacteriophages, and the assembly process. The six N-terminal amino acid residues adopt an unusual "double hook" conformation on the outside of the bacteriophage particle. The solid-state NMR results indicate that at 30 degrees C, some of the coat protein subunits assume a single, fully structured conformation, and some have a few mobile residues that provide a break between two helical segments, in agreement with structural models from X-ray fiber and neutron diffraction, respectively. The atomic resolution structure determined by solid-state NMR for residues 7-14 and 18-46, which excludes the N-terminal double hook and the break between the helical segments, but encompasses more than 80% of the backbone including the distinct kink at residue 29, agrees with that determined by X-ray fiber diffraction with an RMSD value of 2.0 A. The symmetry and distance constraints determined by X-ray fiber and neutron diffraction enable the construction of an accurate model of the bacteriophage particle from the coordinates of the coat protein monomers.     

Determinants that target the integrase of phage HK022 into the mammalian nucleus

The integrase (Int) protein of coliphage HK022 catalyzes the site-specific integration and excision of the phage into and from its Escherichia coli host chromosome. Int expressed from a plasmid in COS1 monkey cells is localized in the nucleus, as is a fusion protein between Int and the green fluorescent protein (GFP). Mutation analysis of the GFP-Int fusion has revealed in Int two regions of positively charged amino acid residues that cooperate in the nuclear localization. One region harbors residues Arg90 and Arg93. The other, which spans residues 307-340 belongs to the catalytic domain of Int, is rich in basic residues and is strongly conserved within the Int protein family. Being localized in the nucleus renders Int of HK022 as a potential recombinase for site-specific gene manipulations in mammals.     

The solution structure of bacteriophage lambda protein W, a small morphogenetic protein possessing a novel fold

Protein W (gpW) from bacteriophage lambda is required for the stabilization of DNA within the phage head and for attachment of tails onto the head during morphogenesis. Although comprised of only 68 residues, it likely interacts with at least two other proteins in the mature phage and with DNA. Thus, gpW is an intriguing subject for detailed structural studies. We have determined its solution structure using NMR spectroscopy and have found it to possesses a novel fold consisting of two alpha-helices and a single two-stranded beta-sheet arranged around a well-packed hydrophobic core. The 14 C-terminal residues of gpW, which are essential for function, are unstructured in solution.     

Genome comparison of Pseudomonas aeruginosa large phages

Pseudomonas aeruginosa phage EL is a dsDNA phage related to the giant phiKZ-like Myoviridae. The EL genome sequence comprises 211,215 bp and has 201 predicted open reading frames (ORFs). The EL genome does not share DNA sequence homology with other viruses and micro-organisms sequenced to date. However, one-third of the predicted EL gene products (gps) shares similarity (Blast alignments of 17-55% amino acid identity) with phiKZ proteins. Comparative EL and phiKZ genomics reveals that these giant phages are an example of substantially diverged genetic mosaics. Based on the position of similar EL and phiKZ predicted gene products, five genome regions can be delineated in EL, four of which are relatively conserved between EL and phiKZ. Region IV, a 17.7 kb genome region with 28 predicted ORFs, is unique to EL. Fourteen EL ORFs have been assigned a putative function based on protein similarity. Assigned proteins are involved in DNA replication and nucleotide metabolism (NAD+-dependent DNA ligase, ribonuclease HI, helicase, thymidylate kinase), host lysis and particle structure. EL-gp146 is the first chaperonin GroEL sequence identified in a viral genome. Besides a putative transposase, EL harbours predicted mobile endonucleases related to H-N-H and LAGLIDADG homing endonucleases associated with group I intron and intein intervening sequences.     

Crystal structure of the coat protein from the GA bacteriophage: model of the unassembled dimer.

There are four groups of RNA bacteriophages with distinct antigenic and physicochemical properties due to differences in surface residues of the viral coat proteins. Coat proteins also play a role as translational repressor during the viral life cycle, binding an RNA hairpin within the genome. In this study, the first crystal structure of the coat protein from a Group II phage GA is reported and compared to the Group I MS2 coat protein. The structure of the GA dimer was determined at 2.8 A resolution (R-factor = 0.20). The overall folding pattern of the coat protein is similar to the Group I MS2 coat protein in the intact virus (Golmohammadi R, Valegård K, Fridborg K, Liljas L. 1993, J Mol Biol 234:620-639) or as an unassembled dimer (Ni Cz, Syed R, Kodandapani R. Wickersham J, Peabody DS, Ely KR, 1995, Structure 3:255-263). The structures differ in the FG loops and in the first turn of the alpha A helix. GA and MS2 coat proteins differ in sequence at 49 of 129 amino acid residues. Sequence differences that contribute to distinct immunological and physical properties of the proteins are found at the surface of the intact virus in the AB and FG loops. There are six differences in potential RNA contact residues within the RNA-binding site located in an antiparallel beta-sheet across the dimer interface. Three differences involve residues in the center of this concave site: Lys/Arg 83, Ser/Asn 87, and Asp/Glu 89. Residue 87 was shown by molecular genetics to define RNA-binding specificity by GA or MS2 coat protein (Lim F. Spingola M, Peabody DS, 1994, J Biol Chem 269:9006-9010). This sequence difference reflects recognition of the nucleotide at position -5 in the unpaired loop of the translational operators bound by these coat proteins. In GA, the nucleotide at this position is a purine whereas in MS2, it is a pyrimidine.     

Studies related to the head-maturation pathway of bacteriophages T4 and T2: II. Nuclear disruption,protein synthesis and particle formation with the mutant 43−·30−·46−

We describe the aberrant phage multiplication of the triple conditional lethal mutant 43^?(polymerase)· 30^?(ligase)·46^?(exonuclease) of bacteriophage T4D in which phage DNA replication is arrested but some late protein synthesis occurs (33). The nuclear disruption is indistinguishable from wild type. Forty-five empty small and empty large particles are assembled per cell when the multiplicity of infection (m.o.i.) is 100. This number corresponds closely to the 38 phage equivalents of cleaved major head protein determined biochemically. By reducing the m.o.i. the number of observable particles decreases, reaching 1–5 per cell at an m.o.i. of 5(+5). The total synthesis of phage related proteins is not significantly dependant on the m.o.i. The synthesis of late proteins is about 10% of that of wild type at high m.o.i. and decreases with the m.o.i. The different early and late proteins do not show the same relative proportions as in wild type and respond differently to an increased m.o.i. These and other results are discussed with respect to the role of phage DNA in prehead assembly and head maturation.     

Structure of domain III of the blood-stage malaria vaccine candidate, Plasmodium falciparum apical membrane antigen 1 (AMA1)

Apical membrane antigen 1 of the malarial parasite Plasmodium falciparum (Pf AMA1) is a merozoite antigen that is considered a strong candidate for inclusion in a malaria vaccine. Antibodies reacting with disulphide bond-dependent epitopes in AMA1 block invasion of host erythrocytes by P.falciparum merozoites, and we show here that epitopes involving sites of mutations in domain III are targets of inhibitory human antibodies. The solution structure of AMA1 domain III, a 14kDa protein, has been determined using NMR spectroscopy on uniformly 15N and 13C/15N-labelled samples. The structure has a well-defined disulphide-stabilised core region separated by a disordered loop, and both the N and C-terminal regions of the molecule are unstructured. Within the disulphide-stabilised core, residues 443-447 form a turn of helix and residues 495-498 and 503-506 an anti-parallel beta-sheet with a distorted type I beta-turn centred on residues 500-501, producing a beta-hairpin-type structure. The structured region of the molecule includes all three disulphide bonds. The previously unassigned connectivities for two of these bonds could not be established with certainty from the NMR data and structure calculations, but were determined to be C490-C507 and C492-C509 from an antigenic analysis of mutated forms of this domain expressed using phage display. Naturally occurring mutations in domain III that are located far apart in the primary sequence tend to cluster in the region of the disulphide core in the three-dimensional structure of the molecule. The structure shows that nearly all the polymorphic sites have a high level of solvent accessibility, consistent with their location in epitopes recognised by protective antibodies. Even though domain III in solution contains significant regions of disorder in the structure, the disulphide-stabilised core that is structured is clearly an important element of the antigenic surface of AMA1 recognised by protective antibodies.     

The conserved carboxy terminus of the capsid domain of human immunodeficiency virus type 1 gag protein is important for virion assembly and release

The retroviral Gag precursor plays an important role in the assembly of virion particles. The capsid (CA) protein of the Gag molecule makes a major contribution to this process. In the crystal structure of the free CA protein of the human immunodeficiency virus type 1 (HIV-1), 11 residues of the C terminus were found to be unstructured, and to date no information exists on the structure of these residues in the context of the Gag precursor molecule. We performed phylogenetic analysis and demonstrated a high degree of conservation of these 11 amino acids. Deletion of this cluster or introduction of various point mutations into these residues resulted in significant impairment of particle infectivity. In this cluster, two putative structural regions were identified, residues that form a hinge region (353-VGGP-356) and those that contribute to an alpha-helix (357-GHKARVL-363). Overall, mutations in these regions resulted in inhibition of virion production, but mutations in the hinge region demonstrated the most significant reduction. Although all the Gag mutants appeared to have normal Gag-Gag and Gag-RNA interactions, the hinge mutants were characterized by abnormal formation of cytoplasmic Gag complexes. Gag proteins with mutations in the hinge region demonstrated normal membrane association but aberrant rod-like membrane structures. More detailed analysis of these structures in one of the mutants demonstrated abnormal trapped Gag assemblies. These data suggest that the conserved CA C terminus is important for HIV-1 virion assembly and release and define a putative target for drug design geared to inhibit the HIV-1 assembly process.