In vitro scanning saturation mutagenesis of all the specificity determining residues in an antibody binding site.

For the first time, each specificity determining residue (SDR) in the binding site of an antibody has been replaced with every other possible single amino acid substitution, and the resulting mutants analyzed for binding affinity and specificity. The studies were conducted on a variant of the 26-10 antidigoxin single chain Fv (scFv) using in vitro scanning saturation mutagenesis, a new process that allows the high throughput production and characterization of antibody mutants [Burks,E.A., Chen,G., Georgiou,G. and Iverson,B.L. (1997) Proc. Natl Acad. Sci. USA, 94, 412-417]. Single amino acid mutants of 26-10 scFv were identified that modulated specificity in dramatic fashion. The overall plasticity of the antibody binding site with respect to amino acid replacement was also evaluated, revealing that 86% of all mutants retained measurable binding activity. Finally, by analyzing the physical properties of amino acid substitutions with respect to their effect on hapten binding, conclusions were drawn regarding the functional role played by the wild-type residue at each SDR position. The reported results highlight the value of in vitro scanning saturation mutagenesis for engineering antibody binding specificity, for evaluating the plasticity of proteins, and for comprehensive structure-function studies and analysis.     

The isolation and characterization of mutants of the integration host factor (IHF) of Escherichia coli with altered, expanded DNA-binding specificities.

The integration host factor (IHF) of Escherichia coli is a small, basic protein that is required for lambda site-specific recombination and a variety of cellular processes. It is composed of two subunits, alpha and beta, that are encoded by the himA and hip (himD) genes, respectively. IHF is a sequence-specific DNA-binding protein and bends the DNA when it binds. We have used the bacteriophage P22-based challenge phage selection to isolate suppressor mutants with altered, expanded DNA binding specificities. The suppressors were isolated by selecting mutants that recognize variants of the phage lambda H'IHF recognition site. Two of the mutants recognize both the wild-type and a single variant site and contain amino acid substitutions at positions 64 (Pro to Leu) or 65 (Lys to Ser) of the alpha subunit. These substitutions are in a region of the protein that is predicted to contain a flexible arm that interacts with DNA. Three other mutants, which recognize the wild-type and a different variant site, contain amino acid substitutions at position 44 (Glu to Lys, Val or Gly) of the beta subunit. These substitutions are in the middle of a predicted beta-strand of the subunit. We discuss the possible mechanisms of suppression by the mutants in terms of a model of the IHF-DNA complex proposed by Yang and Nash [Cell, 57, 869-880 (1989)].     

Genetic analysis of Escherichia coli integration host factor interactions with its bacteriophage lambda H'' recognition site.

The bacteriophage P22-based challenge phage system was used to study the binding of integration host factor (IHF) to its H' recognition site in the attP region of bacteriophage lambda. We constructed challenge phages that carried H' inserts in both orientations within the P22 Pant promoter, which is required for antirepressor synthesis. We found that IHF repressed expression of Pant from either challenge phage when expressed from an inducible Ptac promoter on a plasmid vector. Mutants containing changes in the H' inserts that decrease or eliminate IHF binding were isolated by selecting challenge phages that could synthesize antirepressor in the presence of IHF. Sequence analysis of 31 mutants showed that most changes were base pair substitutions within the H' insert. Approximately one-half of the mutants contained substitutions that changed base pairs that are part of the IHF consensus binding site; mutants were isolated that contained substitutions at six of the nine base pairs of the consensus site. Other mutants contained changes at base pairs between the two subdeterminants of the H' site, at positions that are not specified in the consensus sequence, and in the dA + dT-rich region that flanks the consensus region of the site. Taken together, these results show that single-base-pair changes at positions outside of the proposed consensus bases can weaken or drastically disrupt IHF binding to the mutated site.     

Contribution of a single heavy chain residue to specificity of an anti-digoxin monoclonal antibody.

Two distinct spontaneous variants of the murine anti-digoxin hybridoma 26-10 were isolated by fluorescence-activated cell sorting for reduced affinity of surface antibody for antigen. Nucleotide and partial amino acid sequencing of the variant antibody variable regions revealed that 1 variant had a single amino acid substitution: Lys for Asn at heavy chain position 35. The second variant antibody had 2 heavy chain substitutions: Tyr for Asn at position 35, and Met for Arg at position 38. Mutagenesis experiments confirmed that the position 35 substitutions were solely responsible for the markedly reduced affinity of both variant antibodies. Several mutants with more conservative position 35 substitutions were engineered to ascertain the contribution of Asn 35 to the binding of digoxin to antibody 26-10. Replacement of Asn with Gln reduced affinity for digoxin 10-fold relative to the wild-type antibody, but maintained wild-type fine specificity for cardiac glycoside analogues. All other substitutions (Val, Thr, Leu, Ala, and Asp) reduced affinity by at least 90-fold and caused distinct shifts in fine specificity. The Ala mutant demonstrated greatly increased relative affinities for 16-acetylated haptens and haptens with a saturated lactone. The X-ray crystal structure of the 26-10 Fab in complex with digoxin (Jeffrey PD et al., 1993, Proc Natl Acad Sci USA 90:10310-10314) reveals that the position 35 Asn contacts hapten and forms hydrogen bonds with 2 other contact residues. The reductions in affinity of the position 35 mutants for digoxin are greater than expected based upon the small hapten contact area provided by the wild-type Asn. We therefore performed molecular modeling experiments which suggested that substitution of Gln or Asp can maintain these hydrogen bonds whereas the other substituted side chains cannot. The altered binding of the Asp mutant may be due to the introduction of a negative charge. The similarities in binding of the wild-type and Gln-mutant antibodies, however, suggest that these hydrogen bonds are important for maintaining the architecture of the binding site and therefore the affinity and specificity of this antibody. The Ala mutant eliminates the wild-type hydrogen bonding, and molecular modeling suggests that the reduced side-chain volume also provides space that can accommodate a congener with a 16-acetyl group or saturated lactone, accounting for the altered fine specificity of this antibody.     

Site-specific mutagenesis at positions 272 and 273 of the Bacillus sp. SAM1606 α-glucosidase to screen mutants with altered specificity for oligosaccharide production by transglucosylation

The Bacillus sp. SAM1606 α-glucosidase catalyzes the transglucosylation of sucrose to produce theanderose (6-O^G-glucosylsucrose) as the major transfer product along with the other di-, tri-, and tetrasaccharides. To obtain an α-glucosidase variant(s) producing theanderose more abundantly, we carried out site-specific mutagenesis studies, in which an amino acid residue (Gly273 or Thr272) near the putative catalytic site (Glu271) of this α-glucosidase was replaced by all other naturally-occurring amino acids. Each mutant, whose concentration was set at 2.6 U/ml (sucrose-hydrolyzing units), was reacted at 60 °C and pH 6.0 with 1.75 M sucrose, and the course of the oligosaccharide production was monitored by HPLC to systematically analyze the effects of amino acid substitutions on the specificity of transglucosylation. The analysis clearly showed site- and residue-dependent differential effects of substitution near the catalytic site on the specificity of oligosaccharide production. For example, mutants with substitution at position 273 by aromatic amino acids or His virtually lost the ability to produce oligosaccharides by transglucosylation. Mutants with substitution at position 272 by amino acids that were bulkier than the wild-type Thr showed enhanced production of tetrasaccharides; whereas, mutants with substitution at position 273 by Lys and Arg exclusively produced disaccharidal transfer products. The highest specificity for theanderose formation (i.e. the highest content of theanderose in the reaction product) was obtained with the T272I mutant, which showed 1.74 times higher productivity (per sucrose-hydrolyzing unit) of theanderose than that of the wild-type enzyme.     

Identification of residues critical for metallo-β-lactamase function by codon randomization and selection

IMP-1 beta-lactamase is a zinc metallo-enzyme encoded by the transferable bla(IMP-1) gene, which confers resistance to virtually all beta-lactam antibiotics including carbapenems. To understand how IMP-1 recognizes and hydrolyzes beta-lactam antibiotics it is important to determine which amino acid residues are critical for catalysis and which residues control substrate specificity. We randomized 27 individual codons in the bla(IMP-1) gene to create libraries that contain all possible amino acid substitutions at residue positions in and near the active site of IMP-1. Mutants from the random libraries were selected for the ability to confer ampicillin resistance to Escherichia coli. Of the positions randomized, >50% do not tolerate amino acid substitutions, suggesting they are essential for IMP-1 function. The remaining positions tolerate amino acid substitutions and may influence the substrate specificity of the enzyme. Interestingly, kinetic studies for one of the functional mutants, Asn233Ala, indicate that an alanine substitution at this position significantly increases catalytic efficiency as compared with the wild-type enzyme.     

Improvement of alkaline lipase from Proteus vulgaris T6 by directed evolution

To expand the functionality of lipase from Proteus vulgaris (PVL) we have used error-prone PCR and DNA shuffling methods to create PVL mutants with improved lipase activity. One desirable mutant with three amino acids substitutions was obtained. The mutated lipase was purified and characterized. The activity of the mutant lipase EF3.3 was 3.5 times higher than that of the wild-type (WT-PVL). The mutational effect is interpreted according to a simulated three-dimensional structure for the mutant lipase. Amino acid substitution at position 102 was determined to be critical for lipase activity, while the residue at positions 197 and 229 had only marginal effect.     

AraC proteins with altered DNA sequence specificity which activate a mutant promoter in Escherichia coli

We examined the recognition of the araBAD promoter by the AraC protein in the Escherichia coli arabinose operon. A mutant promoter, with base substitutions at positions contacted by AraC, was used to isolate suppressor mutations in araC by direct selection. Two hydroxylamine-induced araC mutations were isolated repeatedly; each contained a single amino acid substitution. When tested against a set of base substitution promoter mutants, one revertant, an Arg to His substitution at residue 250, displayed altered base specificity for a single position within the araBAD promoter. The other revertant, a Cys to Tyr substitution at residue 204, did not show consistent base-specific suppression. Neither demonstrated a higher affinity than the wild type protein for the mutant promoter in vitro. Both proteins suppress mutant sequences by a mechanism that does not appear to involve the formation of new net favorable contacts with the mutant base pairs of the promoter.     

Use of monoclonal antibodies to identify four neutralization immunogens on a common cold picornavirus, human rhinovirus 14.

A collection of 35 mouse monoclonal antibodies, raised against human rhinovirus 14 (HRV-14), was used to isolate 62 neutralization-resistant mutants. When cross-tested against the antibodies in a neutralization assay, the mutants fell into four antigenic groups, here called neutralization immunogens: NIm-IA, -IB, -II, and -III. Sequencing the mutant RNA in segments corresponding to serotype-variable regions revealed that the amino acid substitutions segregated into clusters, which correlated exactly with the immunogenic groups (NIm-IA mutants at VP1 amino acid residue 91 or 95; NIm-II mutants at VP2 residue 158, 159, 161, or 162; NIm-III mutants at VP3 residue 72, 75, or 78; and NIm-IB mutants at two sites, either VP1 residue 83 or 85, or residue 138 or 139). Examination of the three-dimensional structure of the virus (M. G. Rossmann, E. Arnold, J. W. Erickson, E. A. Frankenberger, J. P. Griffith, H.-J. Hecht, J. E. Johnson, G. Kamer, M. Luo, A. G. Mosser, R. R. Rueckert, B. Sherry, and G. Vriend, Nature [London], 317:145-153, 1985) revealed that each of the substitution clusters formed a protrusion from the virus surface, and the side chains of the substituted amino acids pointed outward. Moreover, four of the amino acid substitutions, which initially appeared to be anomalous because they were encoded well outside the cluster groups, could be traced to surface positions immediately adjacent to the appropriate viral protrusions. We conclude that three of the four antigens, NIm-IB, -II, and -III, are discontinuous. Thus, the amino acid substitutions in all 62 mutants fell within the proposed immunogenic sites; there was no evidence for alteration of any antigenic site by a distal mutation.     

Desaturation and hydroxylation. Residues 148 and 324 of Arabidopsis FAD2, in addition to substrate chain length, exert a major influence in partitioning of catalytic specificity

Exchanging the identity of amino acids at four key locations within the Arabidopsis thaliana oleate desaturase (FAD2) and the Lesquerella fendleri hydroxylase/desaturase (LFAH) was shown to influence partitioning between desaturation and hydroxylation (Broun, P., Shanklin, J., Whittle, E., and Somerville, C. (1998) Science 282, 1315-1317). We report that four analogous substitutions in the FAD2 sequence by their equivalents from the castor oleate hydroxylase result in hydroxy fatty acid accumulation in A. thaliana to the same levels as for the wild-type castor hydroxylase. We also describe the relative contribution of these substitutions, both individually and in combination, by analyzing the products resulting from their expression in A. thaliana and/or Saccharomyces cerevisiae. Yeast expression showed that M324V, a change reachable by a single point mutation, altered the product distribution approximately 49-fold, and that residue 148 is also a predominant determinant of reaction outcome. Comparison of residues at position 148 of FAD2, LFAH, and the Ricinus oleate hydroxylase prompted us to rationally engineer LFAH-N149I, a variant with approximately 1.9-fold increase in hydroxylation specificity compared with that of wild-type LFAH. Control experiments showed that the wild-type Arabidopsis thaliana FAD2 desaturase has inherent, low level, hydroxylation activity. Further, fatty acid desaturases from different kingdoms and with different regiospecificities exhibit similar intrinsic hydroxylase activity, underscoring fundamental mechanistic similarities between desaturation and hydroxylation. For LFAH mutants the hydroxylation:desaturation ratio is 5-9-fold higher for 18-carbon versus 16-carbon substrates, supporting our hypothesis that substrate positioning in the active site plays a key role in the partitioning of catalytic specificity.     

Integration host factor: putting a twist on protein-DNA recognition

Integration host factor (IHF) is a DNA-bending protein that recognizes its cognate sites through indirect readout. Previous studies have shown that binding of wild-type (WT)-IHF is disrupted by a T to A mutation at the center position of a conserved TTR motif in its binding site, and that substitution of betaGlu44 with Ala prevented IHF from discriminating between A and T at this position. We have determined the crystal structures and relative binding affinities for all combinations of WT-IHF and IHF-betaGlu44Ala bound to the WT and mutant DNAs. Comparison of these structures reveals that DNA twist plays a major role in DNA recognition by IHF, and that this geometric parameter is dependent on the dinucleotide step and not on the bound IHF variant.     

Mutational analysis of the octapeptide sequence motif at the NS1-NS2A cleavage junction of dengue type 4 virus.

We have previously shown that proper processing of dengue type 4 virus NS1 from the NS1-NS2A region of the viral polyprotein requires a hydrophobic N-terminal signal and the downstream NS2A. Results from deletion analysis indicate that a minimum length of eight amino acids at the C terminus of NS1 is required for cleavage at the NS1-NS2A junction. Comparison of this eight-amino-acid sequence with the corresponding sequences of other flaviviruses suggests a consensus cleavage sequence of Met/Leu-Val-Xaa-Ser-Xaa-Val-Xaa-Ala. Site-directed mutagenesis was performed to construct mutants of NS1-NS2A that contained a single amino acid substitution at different positions of the consensus cleavage sequence or at the immediate downstream position. Three to eight different substitutions were made at each position. A total of 50 NS1-NS2A mutants were analyzed for their cleavage efficiency relative to that of the wild-type dengue type 4 virus sequence. As predicted, nearly all substitutions at positions P1, P3, P5, P7, and P8, occupied by conserved amino acids, yielded low levels of cleavage, with the exception that Pro or Ala substituting for Ser (P5) was tolerated. Substitutions of an amino acid at the remaining positions occupied by nonconserved amino acids generally yielded high levels of cleavage. However, some substitutions at nonconserved positions were not tolerated. For example, substitution of Gly or Glu for Gln (P4) and substitution of Val or Glu for Lys (P6) each yielded a low level of cleavage. Overall, these data support the proposed cleavage sequence motif deduced by comparison of sequences among the flaviviruses. This study also showed that in addition to the eight-amino-acid sequence, the amino acid immediately following the NS1-NS2A cleavage site plays a role in cleavage.     

Identification of a second site compensatory mutation in the Fe-protein that allows diazotrophic growth of Azotobacter vinelandii UW97

Azotobacter vinelandii UW97 is defective in nitrogen fixation due to a replacement of serine at position 44 by phenylalanine in the Fe-protein [Pulakat, L., Hausman, B.S., Lei, S. and Gavini, N. (1996) J. Biol. Chem. 271, 1884-1889]. Serine residue 44 is located in a conserved domain that links the nucleotide binding site and the MoFe-protein docking surface of the Fe-protein. Therefore, it is possible that the loss of function by A. vinelandii UW97-Fe-protein may be caused by global conformational disruption or disruption of the conformational change upon MgATP binding. To determine whether it is possible to generate a functional nitrogenase complex via a compensating second site mutation(s) in the Fe-protein, we have attempted to isolate genetic revertants of A. vinelandii UW97 that can grow on nitrogen-free medium. One such revertant, designated A vinelandii BG9, encoded a Fe-protein that retained the Ser44Phe mutation and also had a second mutation that caused the replacement of a lysine at position 170 by glutamic acid. Lysine 170 is highly conserved and is located in a conserved region of the Fe-protein. This region is implicated in stabilizing the MgATP-induced conformation of the Fe-protein and in docking to the MoFe-protein. Further complementation analysis showed that the Fe-protein mutant that retained serine 44 but contained the substitution of lysine at position 170 by glutamic acid was also non-functional. Thus, neither Ser44Phe nor Lys170Glu mutants of Fe-protein were functional; however, the Fe-protein in A. vinelandii BG9 that contained both substitutions could support diazotrophic growth on the strain.     

A single highly mutable catalytic site amino acid is critical for DNA polymerase fidelity

DNA polymerases contain active sites that are structurally superimposable and conserved in amino acid sequence. To probe the biochemical and structure-function relationship of DNA polymerases, a large library (200,000 members) of mutant Thermus aquaticus DNA polymerase I (Taq pol I) was created containing random substitutions within a portion of the dNTP binding site (Motif A; amino acids 605-617), and a fraction of all selected active Taq pol I (291 out of 8000) was tested for base pairing fidelity; seven unique mutants that efficiently misincorporate bases and/or extend mismatched bases were identified and sequenced. These mutants all contain substitutions of one specific amino acid, Ile-614, which forms part of the hydrophobic pocket that binds the base and ribose portions of the incoming nucleotide. Mutant Taq pol Is containing hydrophilic substitution I614K exhibit 10-fold lower base misincorporation fidelity, as well as a high propensity to extend mispairs. In addition, these low fidelity mutants containing hydrophilic substitution for Ile-614 can bypass damaged templates that include an abasic site and vinyl chloride adduct ethenoA. During polymerase chain reaction, Taq pol I mutant I614K exhibits an error rate that is >20-fold higher relative to the wild-type enzyme and efficiently catalyzes both transition and transversion errors. These studies have generated polymerase chain reaction-proficient mutant polymerases containing substitutions within the active site that confers low base pairing fidelity and a high error rate. Considering the structural and sequence conservation of Motif A, it is likely that a similar substitution will yield active low fidelity DNA polymerases that are mutagenic.     

Determining the DNA sequence elements required for binding integration host factor to two different target sites.

Binding sites for the Escherichia coli protein integration host factor (IHF) include a set of conserved bases that can be summarized by the consensus sequence WATCAANNNNTTR (W is dA or dT, R is dA or dG, and N is any nucleotide). However, additional 5'-proximal bases, whose common feature is a high dA+dT content, are also thought to be required for binding at some sites. We examine the relative contribution of these two sequence elements to IHF binding to the H' and H1 sites in attP of bacteriophage lambda by using the bacteriophage P22-based challenge-phage system. IHF was unable to act as a repressor in the challenge-phage assay at H' sites containing the core consensus element but lacking the dA+dT-rich element. This indicates that both elements are required for IHF to bind to the H' site. In contrast, the core consensus determinant alone is sufficient for IHF binding to the H1 site, which lacks an upstream dA+dT-rich region. Fifty mutants that decreased or eliminated IHF binding to the H1 site were isolated. Sequence analysis showed changes in the bases in the core consensus element only, further indicating that this determinant is sufficient for IHF binding to the H1 site. We found that placement of a dA+dT-rich element upstream of the H1 core consensus element significantly increased the affinity, suggesting that the presence of a dA+dT-rich element enhances IHF binding.     

Codon substitution mutations at two positions in the L polymerase protein of human parainfluenza virus type 1 yield viruses with a spectrum of attenuation in vivo and increased phenotypic stability in vitro

The Y942H and L992F temperature-sensitive (ts) and attenuating amino acid substitution mutations, previously identified in the L polymerase of the HPIV3cp45 vaccine candidate, were introduced into homologous positions of the L polymerase of recombinant human parainfluenza virus type 1 (rHPIV1). In rHPIV1, the Y942H mutation specified the ts phenotype in vitro and the attenuation (att) phenotype in hamsters, whereas the L992F mutation specified neither phenotype. Each of these codon mutations was generated by a single nucleotide substitution and therefore had the potential to readily revert to a codon specifying the wild-type amino acid residue. We introduced alternative amino acid assignments at codon 942 or 992 as a strategy to increase genetic stability and to generate mutants that exhibit a range of attenuation. Twenty-three recombinants with codon substitutions at position 942 or 992 of the L protein were viable. One highly ts and att mutant, the Y942A virus, which had a difference of three nucleotides from the codon encoding a wild-type tyrosine, also possessed a high level of genetic and phenotypic stability upon serial passage in vitro at restrictive temperatures compared to that of the parent Y942H virus, which possessed a single nucleotide substitution. We obtained mutants with substitutions at position 992 that, in contrast to the L992F virus, possessed the ts and att phenotypes. These findings identify the use of alternative codon substitution mutations as a method that can be used to generate candidate vaccine viruses with increased genetic stability and/or a modified level of attenuation.     

A single residue in leucyl-tRNA synthetase affecting amino acid specificity and tRNA aminoacylation

Human mitochondrial leucyl-tRNA synthetase (hs mt LeuRS) achieves high aminoacylation fidelity without a functional editing active site, representing a rare example of a class I aminoacyl-tRNA synthetase (aaRS) that does not proofread its products. Previous studies demonstrated that the enzyme achieves high selectivity by using a more specific synthetic active site that is not prone to errors under physiological conditions. Interestingly, the synthetic active site of hs mt LeuRS displays a high degree of homology with prokaryotic, lower eukaryotic, and other mitochondrial LeuRSs that are less specific. However, there is one residue that differs between hs mt and Escherichia coli LeuRSs located on a flexible closing loop near the signature KMSKS motif. Here we describe studies indicating that this particular residue (K600 in hs mt LeuRS and L570 in E. coli LeuRS) strongly impacts aminoacylation in two ways: it affects both amino acid discrimination and transfer RNA (tRNA) binding. While this residue may not be in direct contact with the amino acid or tRNA substrate, substitutions of this position in both enzymes lead to altered catalytic efficiency and perturbations to the discrimination of leucine and isoleucine. In addition, tRNA recognition and aminoacylation is affected. These findings indicate that the conformation of the synthetic active site, modulated by this residue, may be coupled to specificity and provide new insights into the origins of selectivity without editing.     

Effect of point mutations in the native simian virus 40 tumor antigen, and in synthetic peptides corresponding to the H-2Db-restricted epitopes, on antigen presentation and recognition by cytotoxic T lymphocyte clones.

The effects on CTL recognition of individual amino acid substitutions within epitopes I, II, and III of SV40 tumor Ag (T Ag) were examined. Epitope I spans amino acids 207 to 215, and epitope II/III is within residues 223 to 231 of SV40 T Ag. An amino acid substitution at position 207 (Ala----Val) or 214 (Lys----Glu) of SV40 T Ag expressed in transformed cells resulted in loss of epitope I, recognized by CTL clone Y-1. The amino acid substitution at residue 214 in the corresponding synthetic peptide, LT207-215(214-Lys----Glu), also led to loss of recognition by CTL clone Y-1. The recognition, by CTL clone Y-1, of peptides LT207-215 and LT207-217 with an Ala----Val substitution at position 207 was severely affected. Peptides LT205-215 and LT205-219 with the Ala----Val substitution at residue 207 were, however, recognized by CTL clone Y-1, suggesting that residues 205 and 206 may be involved in presentation of site I. Alteration of residue 224 (Lys----Glu) in the native T Ag resulted in loss of recognition by both CTL clones Y-2 and Y-3. However, a peptide corresponding to epitope II/III with an identical amino acid substitution at residue 224 provided a target for CTL clone Y-3 but not clone Y-2. A change of Lys----Gln at residue 224 in both the native protein and a synthetic peptide caused loss of recognition by CTL clone Y-2 but not CTL clone Y-3. Further, an amino acid substitution of Lys----Arg at position 224 of the native T Ag decreased recognition of epitope II/III by CTL clones Y-2 and Y-3 but had no effect on recognition of a synthetic peptide bearing the same substitution. These results indicate that the mutagenesis approach, resulting in identical amino acid substitutions in the native protein and in the synthetic peptides, may provide insight into the role of individual residues in the processing, presentation, and recognition of CTL recognition epitopes.     

Crystal structures of two mutant neuraminidase-antibody complexes with amino acid substitutions in the interface.

The site on influenza virus N9 neuraminidase recognized by NC41 monoclonal antibody comprises 19 amino acid residues that are in direct contact with 17 residues on the antibody. Single sequence changes in some of the neuraminidase residues in the site markedly reduce antibody binding. However, two mutants have been found within the site, Ile368 to Arg and Asn329 to Asp selected by antibodies other than NC41, and these mutants bind NC41 antibody with only slightly reduced affinity. The three-dimensional structures of the two mutant N9-NC41 antibody complexes as derived from the wild-type complex are presented. Both structures show that some amino acid substitutions can be accommodated within an antigen-antibody interface by local structural rearrangements around the mutation site. In the Ile368 to Arg mutant complex, the side-chain of Arg368 is shifted by 2.9 A from its position in the uncomplexed mutant and a shift of 1.3 A in the position of the light chain residue HisL55 with respect to the wild-type complex is also observed. In the other mutant, the side-chain of Asp329 appears rotated by 150 degrees around C alpha-C beta with respect to the uncomplexed mutant, so that the carboxylate group is moved to the periphery of the antigen-antibody interface. The results provide a basis for understanding some of the potential structural effects of somatic hypermutation on antigen-antibody binding in those cases where the mutation in the antibody occurs at antigen-contacting residues, and demonstrate again the importance of structural context in evaluating the effect of amino acid substitutions on protein structure and function.     

Determinants of sequence-specific DNA methylation: target recognition and catalysis are coupled in M.HhaI

Sequence specificity studies of the wild-type bacterial DNA cytosine C5 methyltransferase HhaI were carried out with cognate (5'GCGC3') and noncognate DNA substrates containing single base pair changes at the first and the fourth position (underlined). Specificity for noncognate site methylation at the level of kcat/KDDNA is decreased 9000-80000-fold relative to the cognate site, manifested through changes in methylation, or a prior step, and changes in KDDNA. Analysis of a new high-resolution enzyme-DNA cocrystal structure provides a partial mechanistic understanding of this discrimination. To probe the significance of conformational transitions occurring prior to catalysis in determining specificity, we analyzed the double mutant (H127A/T132A). These amino acid substitutions disrupt the interface between the flexible loop (residues 80-99), which interacts with the DNA minor groove, and the active site. The mutant's methylation of the cognate site is essentially unchanged, yet its methylation of noncognate sites is decreased up to 460-fold relative to the wild-type enzyme. We suggest that a significant contribution to M.HhaI's specificity involves the stabilization of reaction intermediates prior to methyl transfer, mediated by DNA minor groove-protein flexible loop interactions.