Site-specific reflex response of ubiquitin to loop insertions

Predicting the structural effects of insertions in proteins by homology modeling remains a challenge. To investigate the molecular basis for conformational adaptations to insertions, ten mutants of ubiquitin were generated by introducing five different inserts, varying from five to 11 residues in size, at two different sites. Most insertion sequences were derived from homologous positions in structurally homologous ubiquitin-like proteins; to test sequence specificity, insertions were made into both homologous and non-homologous sites in ubiquitin. Structural inferences from NMR data suggest that each insertion site shows a reflex response to insertions: the sequence of the insertion has much less impact on structural adaptations than does the site of the insertion. Further, each site responds to insertions in a unique but consistent manner. For a given insertion site, different inserted sequences give rise to different stabilities, but the relationship between stability and sequence is not yet clear. However, the change in stability is similar for all insertions in a given site.     

Predicting the magnitude of the reflex response to insertions in ubiquitin

The ability to predict the structural response of a protein to an insertion would be a significant advance for the fields of homology modeling and protein design. However, the effects of insertions on protein conformation are not well understood. Previous work has demonstrated that for two loops in ubiquitin, the primary determinant of the structural adaptation to insertions is the insertion site rather than the sequence of the insertion; this phenomenon was termed the reflex response of loops to insertions. We report herein the analysis of ubiquitin mutants with insertions in two other loops. This study demonstrates that the insertion site is the primary determinant of the response to insertions for these two new loops as well, which further supports the reflex response hypothesis. We also attempted to predict the relative magnitudes of the responses at each site but were unsuccessful. Using the additional data collected in this work, we have refined our predictive hypothesis.     

Importance of secondary structural specificity determinants in protein folding: insertion of a native beta-sheet sequence into an alpha-helical coiled-coil

To examine how a short secondary structural element derived from a native protein folds when in a different protein environment, we inserted an 11-residue beta-sheet segment (cassette) from human immunoglobulin fold, Fab new, into an alpha-helical coiled-coil host protein (cassette holder). This de novo design protein model, the structural cassette mutagenesis (SCM) model, allows us to study protein folding principles involving both short- and long-range interactions that affect secondary structure stability and conformation. In this study, we address whether the insertion of this beta-sheet cassette into the alpha-helical coiled-coil protein would result in conformational change nucleated by the long-range tertiary stabilization of the coiled-coil, therefore overriding the local propensity of the cassette to form beta-sheet, observed in its native immunoglobulin fold. The results showed that not only did the nucleating helices of the coiled-coil on either end of the cassette fail to nucleate the beta-sheet cassette to fold with an alpha-helical conformation, but also the entire chimeric protein became a random coil. We identified two determinants in this cassette that prevented coiled-coil formation: (1) a tandem dipeptide NN motif at the N-terminal of the beta-sheet cassette, and (2) the hydrophilic Ser residue, which would be buried in the hydrophobic core if the coiled-coil structure were to fold. By amino acid substitution of these helix disruptive residues, that is, either the replacement of the NN motif with high helical propensity Ala residues or the substitution of Ser with Leu to enhance hydrophobicity, we were able to convert the random coil chimeric protein into a fully folded alpha-helical coiled-coil. We hypothesized that this NN motif is a "secondary structural specificity determinant" which is very selective for one type of secondary structure and may prevent neighboring residues from adopting an alternate protein fold. These sequences with secondary structural specificity determinants have very strong local propensity to fold into a specific secondary structure and may affect overall protein folding by acting as a folding initiation site.     

Accommodation of insertion mutations on the surface and in the interior of staphylococcal nuclease.

Alignment of homologous amino acid sequences reveals that insertion mutations are fairly common in evolution. Hitherto, the structural consequences of insertion mutations on the surface and in the interior of proteins of known structures have received little attention. We report here the high-resolution X-ray crystal structures of 2 site-directed insertion mutants of staphylococcal nuclease. The structure of the first insertion mutant, in which 2 glycine residues were inserted on the protein surface in the amino-terminal beta-strand, has been solved to 1.70 A resolution and refined to a crystallographic R value of 0.182. The inserted residues are accommodated in a special 3-residue beta-bulge. A bridging water molecule in the newly created cavity satisfies the hydrogen bonding requirements of the beta-sheet by forming a bifurcated hydrogen bond to 1 beta-strand, and a single hydrogen bond to the other beta-strand. The second insertion mutant contains a single leucine residue inserted at the end of the third beta-strand. The structure was solved to 2.0 A resolution and refined to a final R value of 0.196. The insertion is accommodated in a register shift that changes the conformation of the flexible loop portion of the molecule, relaxing and widening the omega turn. This structural alteration results in changes in position and coordination of a bound calcium ion important for catalysis. These structures illustrate important differences in how amino acid insertions are accommodated: as localized bulges, and as extensive register shifts.     

Fold change in evolution of protein structures

Typically, protein spatial structures are more conserved in evolution than amino acid sequences. However, the recent explosion of sequence and structure information accompanied by the development of powerful computational methods led to the accumulation of examples of homologous proteins with globally distinct structures. Significant sequence conservation, local structural resemblance, and functional similarity strongly indicate evolutionary relationships between these proteins despite pronounced structural differences at the fold level. Several mechanisms such as insertions/deletions/substitutions, circular permutations, and rearrangements in beta-sheet topologies account for the majority of detected structural irregularities. The existence of evolutionarily related proteins that possess different folds brings new challenges to the homology modeling techniques and the structure classification strategies and offers new opportunities for protein design in experimental studies.     

Structures of ubiquitin insertion mutants support site-specific reflex response to insertions hypothesis

We previously concluded that, judging from NMR chemical shifts, the effects of insertions into ubiquitin on its conformation appear to depend primarily on the site of insertion rather than the sequence of the insertion. To obtain a more complete and atomic-resolution understanding of how these insertions modulate the conformation of ubiquitin, we have solved the crystal structures of four insertional mutants of ubiquitin. Insertions between residues 9 and 10 of ubiquitin are minimally perturbing to the remainder of the protein, while larger alterations occur when the insertion is between residues 35 and 36. Further, the alterations in response to insertions are very similar for each mutant at a given site. Two insertions, one at each site, were designed from structurally homologous proteins. Interestingly, the secondary structure within these five to seven amino acid residue insertions is conserved in the new protein. Overall, the crystal structures support the previous conclusion that the conformational effects of these insertions are determined largely by the site of insertion and only secondarily by the sequence of the insert.     

Active TEM-1 beta-lactamase mutants with random peptides inserted in three contiguous surface loops

Engineering of alternative binding sites on the surface of an enzyme while preserving the enzymatic activity would offer new opportunities for controlling the activity by binding of non-natural ligands. Loops and turns are the natural substructures in which binding sites might be engineered with this purpose. We have genetically inserted random peptide sequences into three relatively rigid and contiguous loops of the TEM-1 beta-lactamase and assessed the tolerance to insertion by the percentage of active mutants. Our results indicate that tolerance to insertion could not be correlated to tolerance to mutagenesis. A turn between two beta-strands bordering the active site was observed to be tolerant to random mutagenesis but not to insertions. Two rigid loops comprising rather well-conserved amino acid residues tolerated insertions, although with some constraints. Insertions between the N-terminal helix and the first beta-strand generated active libraries if cysteine residues were included at both ends of the insert, suggesting the requirement for a stabilizing disulfide bridge. Random sequences were relatively well accommodated within the loop connecting the final beta-strand to the C-terminal helix, particularly if the wild-type residue was retained at one of the loops' end. This suggests two strategies for increasing the percentage of active mutants in insertion libraries. The amino acid distribution in the engineered loops was analyzed and found to be less biased against hydrophobic residues than in natural medium-sized loops. The combination of these activity-selected libraries generated a huge library containing active hybrid enzymes with all three loops modified.     

Prediction and identification of a permissive epitope insertion site in the vesicular stomatitis virus glycoprotein

We developed a rational approach to identify a site in the vesicular stomatitis virus (VSV) glycoprotein (G) that is exposed on the protein surface and tolerant of foreign epitope insertion. The foreign epitope inserted was the six-amino-acid sequence ELDKWA, a sequence in a neutralizing epitope from human immunodeficiency virus type 1. This sequence was inserted into six sites within the VSV G protein (Indiana serotype). Four sites were selected based on hydrophilicity and high sequence variability identified by sequence comparison with other vesiculovirus G proteins. The site showing the highest variability was fully tolerant of the foreign peptide insertion. G protein containing the insertion at this site folded correctly, was transported normally to the cell surface, had normal membrane fusion activity, and could reconstitute fully infectious VSV. The virus was neutralized by the human 2F5 monoclonal antibody that binds the ELDKWA epitope. Additional studies showed that this site in G protein tolerated insertion of at least 16 amino acids while retaining full infectivity. The three other insertions in somewhat less variable sequences interfered with VSV G folding and transport to the cell surface. Two additional insertions were made in a conserved sequence adjacent to a glycosylation site and near the transmembrane domain. The former blocked G-protein transport, while the latter allowed transport to the cell surface but blocked membrane fusion activity of G protein. Identification of an insertion-tolerant site in VSV G could be important in future vaccine and targeting studies, and the general principle might also be useful in other systems.     

NMR Structure of the Amino-Terminal Domain of the Lambda Integrase Protein in Complex with DNA: Immobilization of a Flexible Tail Facilitates Beta-Sheet Recognition of the Major Groove

The integrase protein (Int) from bacteriophage lambda is the archetypal member of the tyrosine recombinase family, a large group of enzymes that rearrange DNA in all domains of life. Int catalyzes the insertion and excision of the viral genome into and out of the Escherichia coli chromosome. Recombination transpires within higher-order nucleoprotein complexes that form when its amino-terminal domain binds to arm-type DNA sequences that are located distal to the site of strand exchange. Arm-site binding by Int is essential for catalysis, as it promotes Int-mediated bridge structures that stabilize the recombination machinery. We have elucidated how Int is able to sequence specifically recognize the arm-type site sequence by determining the solution structure of its amino-terminal domain (Int^N, residues Met1 to Leu64) in complex with its P′2 DNA binding site. Previous studies have shown that Int^N adopts a rare monomeric DNA binding fold that consists of a three-stranded antiparallel beta-sheet that is packed against a carboxy-terminal alpha helix. A low-resolution crystal structure of the full-length protein also revealed that the sheet is inserted into the major groove of the arm-type site. The solution structure presented here reveals how Int^N specifically recognizes the arm-type site sequence. A novel feature of the new solution structure is the use of an 11-residue tail that is located at the amino terminus. DNA binding induces the folding of a 3₁₀ helix in the tail that projects the amino terminus of the protein deep into the minor groove for stabilizing DNA contacts. This finding reveals the structural basis for the observation that the “unstructured” amino terminus is required for recombination.     

Homology modelling and protein engineering strategy of subtilases, the family of subtilisin-like serine proteinases.

Subtilases are members of the family of subtilisin-like serine proteases. Presently, greater than 50 subtilases are known, greater than 40 of which with their complete amino acid sequences. We have compared these sequences and the available three-dimensional structures (subtilisin BPN', subtilisin Carlsberg, thermitase and proteinase K). The mature enzymes contain up to 1775 residues, with N-terminal catalytic domains ranging from 268 to 511 residues, and signal and/or activation-peptides ranging from 27 to 280 residues. Several members contain C-terminal extensions, relative to the subtilisins, which display additional properties such as sequence repeats, processing sites and membrane anchor segments. Multiple sequence alignment of the N-terminal catalytic domains allows the definition of two main classes of subtilases. A structurally conserved framework of 191 core residues has been defined from a comparison of the four known three-dimensional structures. Eighteen of these core residues are highly conserved, nine of which are glycines. While the alpha-helix and beta-sheet secondary structure elements show considerable sequence homology, this is less so for peptide loops that connect the core secondary structure elements. These loops can vary in length by greater than 150 residues. While the core three-dimensional structure is conserved, insertions and deletions are preferentially confined to surface loops. From the known three-dimensional structures various predictions are made for the other subtilases concerning essential conserved residues, allowable amino acid substitutions, disulphide bonds, Ca(2+)-binding sites, substrate-binding site residues, ionic and aromatic interactions, proteolytically susceptible surface loops, etc. These predictions form a basis for protein engineering of members of the subtilase family, for which no three-dimensional structure is known.     

Structural diversity of domain superfamilies in the CATH database

The CATH database of domain structures has been used to explore the structural variation of homologous domains in 294 well populated domain structure superfamilies, each containing at least three sequence diverse relatives. Our analyses confirm some previously detected trends relating sequence divergence to structural variation but for a much larger dataset and in some superfamilies the new data reveal exceptional structural variation. Use of a new algorithm (2DSEC) to analyse variability in secondary structure compositions across a superfamily sheds new light on how structures evolve. 2DSEC detects inserted secondary structures that embellish the core of conserved secondary structures found throughout the superfamily. Analysis showed that for 56% of highly populated superfamilies (>9 sequence diverse relatives), there are twofold or more increases in the numbers of secondary structures in some relatives. In some families fivefold increases occur, sometimes modifying the fold of the domain. Manual inspection of secondary structure insertions or embellishments in 48 particularly variable superfamilies revealed that although these insertions were usually discontiguous in the sequence they were often co-located in 3D resulting in a larger structural motif that often modified the geometry of the active site or the surface conformation promoting diverse domain partnerships and protein interactions. These observations, supported by automatic analysis of all well populated CATH families, suggest that accretion of small secondary structure insertions may provide a simple mechanism for evolving new functions in diverse relatives. Some layered domain architectures (e.g. mainly-beta and alpha-beta sandwiches) that recur highly in the genomes more frequently exploit these types of embellishments to modify function. In these architectures, aggregation occurs most often at the edges, top or bottom of the beta-sheets. Information on structural variability across domain superfamilies has been made available through the CATH Dictionary of Homologous Structures (DHS).     

Low free energy cost of very long loop insertions in proteins

Long insertions into a loop of a folded host protein are expected to have destabilizing effects because of the entropic cost associated with loop closure unless the inserted sequence adopts a folded structure with amino- and carboxy-termini in close proximity. A loop entropy reduction screen based on this concept was used in an attempt to retrieve folded sequences from random sequence libraries. A library of long random sequences was inserted into a loop of the SH2 domain, displayed on the surface of M13 phage, and the inserted sequences that did not disrupt SH2 function were retrieved by panning using beads coated with a phosphotyrosine containing SH2 peptide ligand. Two sequences of a library of 2 x 10(8) sequences were isolated after multiple rounds of panning, and were found to have recovery levels similar to the wild-type SH2 domain and to be relatively intolerant to further mutation in PCR mutagenesis experiments. Surprisingly, although these inserted sequences exhibited little nonrandom structure, they do not significantly destabilize the host SH2 domain. Additional insertion variants recovered at lower levels in the panning experiments were also found to have a minimal effect on the stability and peptide-binding function of the SH2 domain. The additional level of selection present in the panning experiments is likely to involve in vivo folding and assembly, as there was a rough correlation between recovery levels in the phage-panning experiments and protein solubility. The finding that loop insertions of 60-80 amino acids have minimal effects on SH2 domain stability suggests that the free energy cost of inserting long loops may be considerably less than polymer theory estimates based on the entropic cost of loop closure, and, hence, that loop insertion may have provided an evolutionary route to multidomain protein structures.     

Ribosomal DNA in Drosophila melanogaster. II. Heteroduplex mapping of cloned and uncloned rDNA.

Sequences in the cloned Drosophila melanogaster rDNA fragments described by Dawid et al. (1978) were compared by heteroduplex mapping. The nontranscribed spacer regions in all fragments are homologous but vary in length. Deletion loops were observed at variable positions in the spacer region suggesting that spacers are internally repetitious.Many rDNA repeats in D. melanogaster have a 28 S gene interrupted by a region named the ribosomal insertion. Insertions of 0.5, 1 and 5 kb were found in repeat-length EcoRI fragments. These DNA regions, named type 1 insertions, are homologous at their right ends. Although 1 kb insertions are quite precisely twice as large as 0.5 kb insertions they do not represent a duplication of the shorter sequence. Some insertions have at least one EcoRI site and therefore yield EcoRI fragments which are only part of a repeat. The sequences in two cloned right-hand partial insertion sequences are homologous, but the sequences in two lefthand partial insertions are not. None of the EcoRI-restrictable insertion sequences has any homology to any part of type 1 insertions; they are thus grouped together as type 2. Evidence for insertion sequences of at least two types in uncloned rDNA was obtained by annealing a cloned fragment with a 1 kb insertion to genomic rDNA. About 15% of the rDNA repeats show substitution type loops between the 1 kb type 1 insertion derived from the cloned fragment and type 2 insertions in the rDNA.     

Transposable elements associated with constitutive expression of yeast alcohol dehydrogenase II

The yeast structural gene ADR2, coding for the glucose-repressible alcohol dehydrogenase (ADHII), has been isolated by complementation of function in transformed yeast. The chromosomal DNA from nine yeast strains with cis-dominant constitutive mutations (ADR3^c) has been investigated by restriction enzyme analysis, using the cloned ADR2 DNA as a hybridization probe. Seven mutants appear to have insertions of approximately 5.6 kb near the 5′ end of the ADR2-coding region. Four of these insertions have the same restriction pattern as the yeast transposable element Tyl. Two differ from Tyl by the presence of an additional Hind III site, and a seventh insertion differs from Tyl at a number of restriction sites. All are inserted in the same orientation with respect to the structural gene. A DNA fragment containing the ADR2 gene and adjacent sequences from a constitutive mutant has been cloned and shown by heteroduplex analysis to contain an insertion near the 5′ end of the structural gene. The cloned insertion sequence hybridizes to multiple genomic DNA fragments, indicating that it contains a moderately repetitive sequence. Thus it appears that insertion of a transposable element near the 5′ terminus of the structural gene can produce constitutive expression of a normally glucose-repressed enzyme. Such insertions seem to be the most common way of generating cis-dominant constitutive mutations of ADHII.     

IS1 insertion generates duplication of a nine base pair sequence at its target site.

Three independent integrations of the E. coli insertion sequence, IS1, into the gal operon have been analyzed. DNA sequences of portions of the wild-type galT gene which act as the target sites for these insertions, as well as the corresponding gal/IS1 junctions, are reported. Two features are particularly noteworthy. First, similar sequences appearing in inverted orientation consitute the ends of IS1: 18 of the terminal 23 base pairs at each end are identical. Second, in all three insertions, a 9 base pair segment found once in the wild-type sequence at the site of insertion is duplicated and appears in the same orientation at each end of the inserted element. The sequence of this 9 base pair repeat is different for each insertion analyzed. No homology between the inverted repeat sequences at the ends of IS1 and the sequences of the target sites is observed. Models for the mechanism of IS1 insertion are proposed.     

Functionally accepted insertions of proteins within protein domains

Experiments were designed to explore the tolerance of protein structure and folding to very large insertions of folded protein within a structural domain. Dihydrofolate reductase and beta-lactamase have been inserted in four different positions of phosphoglycerate kinase. The resultant chimeric proteins are all overexpressed, and the host as well as the inserted partners are functional. Although not explicitly designed, functional coupling between the two fused partners was observed in some of the chimeras. These results show that the tolerance of protein structures to very large structured insertions is more general than previously expected and supports the idea that the natural sequence continuity of a structural domain is not required for the folding process. These results directly suggest a new experimental approach to screen, for example, for folded protein in randomized polypeptide sequences.     

Electron microscope analysis of mouse and rabbit globin and immunoglobulin gene sequences

Mouse and rabbit globin and immunoglobulin gene sequences, which had been synthesized in vitro from eukaryotic mRNAs and inserted into plasmids, have been examined in the electron microscope. The size of the inserted β rabbit and α and β mouse globin DNA sequences has been estimated as 620 base pairs while the size of the inserted α rabbit globin DNA sequences was found to be about 490 base pairs. Heteroduplex analysis has revealed no structural abnormalities at the insertion sites of the chimeric plasmids except in the case of a plasmid containing an immunoglobulin light chain gene sequence of about 830 bases, in which a 3 kb deletion adjacent to the insertion site was observed.     

Insertion and excision of Bacteroides conjugative chromosomal elements.

Many strains of Bacteroides harbor large chromosomal elements that can transfer themselves from the chromosome of the donor to the chromosome of the recipient. Most of them carry a tetracycline resistance (Tcr) gene and have thus been designated Tcr elements. In the present study, we have used transverse alternating field electrophoresis to show that all but one of the Tcr elements screened were approximately 70 to 80 kbp in size. The exception (Tcr Emr 12256) was 150 to 200 kbp in size and may be a hybrid element. All of the Tcr elements inserted in more than one site, but insertion was not random. The Tcr elements sometimes cotransfer unlinked chromosomal segments, or nonreplicating Bacteroides units (NBUs). Transverse alternating field electrophoresis analysis showed that insertion of NBUs was not random and that the NBUs did not insert near the Tcr element. Although attempts to clone one or both ends of a Tcr element have not been successful, ends of a cryptic element (XBU4422) were cloned previously and shown to be homologous to the ends of Tcr elements. We have obtained DNA sequences of junction regions between XBU4422 and its target from several different insertions. Comparison of junction sequences with target sequences showed that no target site duplication occurred during insertion and that XBU4422 carried 4 to 5 bp of adjacent chromosomal DNA when it excised from the chromosome and inserted in a plasmid. We identified a short region of sequence similarity between one of the ends of XBU4422 and its target site that may be important for insertion. This sequence contained an 8-bp segment that was identical to the recombinational hot spot sequence on Tn21. XBU4422 could exise itself from plasmids into which it inserted. In most cases, the excision left a single additional A behind in the target site, but precise excision was seen in one case.     

Structural Alphabets for Protein Structure Classification: A Comparison Study

Finding structural similarities between proteins often helps reveal shared functionality, which otherwise might not be detected by native sequence information alone. Such similarity is usually detected and quantified by protein structure alignment. Determining the optimal alignment between two protein structures, however, remains a hard problem. An alternative approach is to approximate each three-dimensional protein structure using a sequence of motifs derived from a structural alphabet. Using this approach, structure comparison is performed by comparing the corresponding motif sequences or structural sequences. In this article, we measure the performance of such alphabets in the context of the protein structure classification problem. We consider both local and global structural sequences. Each letter of a local structural sequence corresponds to the best matching fragment to the corresponding local segment of the protein structure. The global structural sequence is designed to generate the best possible complete chain that matches the full protein structure. We use an alphabet of 20 letters, corresponding to a library of 20 motifs or protein fragments having four residues. We show that the global structural sequences approximate well the native structures of proteins, with an average coordinate root mean square of 0.69 Å over 2225 test proteins. The approximation is best for all α-proteins, while relatively poorer for all β-proteins. We then test the performance of four different sequence representations of proteins (their native sequence, the sequence of their secondary-structure elements, and the local and global structural sequences based on our fragment library) with different classifiers in their ability to classify proteins that belong to five distinct folds of CATH. Without surprise, the primary sequence alone performs poorly as a structure classifier. We show that addition of either secondary-structure information or local information from the structural sequence considerably improves the classification accuracy. The two fragment-based sequences perform better than the secondary-structure sequence but not well enough at this stage to be a viable alternative to more computationally intensive methods based on protein structure alignment.