Hydrogen exchange/electrospray ionization mass spectrometry studies of structural features of proteins and protein/protein interactions

The rate at which amide hydrogens located at the peptide backbone in protein/protein complexes undergo hydrogen/deuterium exchange is highly dependent on whether the amide groups participate in binding. Here, a new mass spectrometric method is presented in which this effect is utilized for the characterization of protein/ligand binding sites. The information obtained is which region within the protein participates in binding. The method includes hydrogen/deuterium exchange of receptor and ligand protein amide protons, binding, and back exchange. After this procedure those backbone amide groups that participate in protein binding are protected from back exchange and therefore still deuterated. These regions were then identified by peptic proteolysis, fast microbore high-performance liquid chromatography separation, and electrospray ionization mass spectrometry. The approach has been applied to the investigation of structural features of insulin-like growth factor I (IGF-I) and the interaction of insulin-like growth factor I with IGF-I binding protein 1. The data show that the approach can provide information on the location of the hydrophobic core of IGF-1 and on two regions that are mainly involved in binding to IGF-I binding protein 1. The data are consistent with results obtained with other approaches. The amount of sample required for one experiment is in the subnanomolar range.     

Stopped-flow NMR measurement of hydrogen exchange rates in reduced horse cytochrome c under strongly destabilizing conditions

A procedure to measure exchange rates of fast exchanging protein amide hydrogens by time-resolved NMR spectroscopy following in situ initiation of the reaction by diluting a native protein solution into an exchanging deuterated buffer is described. The method has been used to measure exchange rates of a small set of amide hydrogens of reduced cytochrome c, maintained in a strictly anaerobic atmosphere, in the presence of an otherwise inaccessible range of guanidinium deuterochloride concentrations. The results for the measured protons indicate that hydrogen exchange in the unfolding transition region of cytochrome c reach the EX2 limit, but emphasize the difficulty in interpretation of the exchange mechanism in protein hydrogen exchange studies. Comparison of free energies of structure opening for the measured hydrogens with the global unfolding free energy monitored by far-UV CD measurements has indicated the presence of at least one partially unfolded equilibrium species of reduced cytochrome c. The results provide the first report of measurement of free energy of opening of structure to exchange in the 0–2-kcal/mol range. Proteins 32:241–247, 1998. © 1998 Wiley-Liss, Inc.     

Epitope localization in antigen-monoclonal-antibody complexes by small-angle X-ray scattering. An approach to domain organization in the beta 2 subunit of Escherichia coli tryptophan synthase

Each polypeptide chain of the beta 2 subunit of Escherichia coli tryptophan synthase (EC 4.2.1.20) is made of two domains, F1 (N-terminal) and F2 (C-terminal). To determine the relative position of these domains in the native protein, complexes between beta 2 and Fab fragments from two monoclonal antibodies, one specific for F1 (68-1) and the other for F2 (93-6), have been prepared and purified. Small-angle X-ray scattering measurements have been made on solutions of each complex. From the experimental scattering curves obtained, computer modeling leads to structural models of the two beta 2-Fab complexes. Though relatively low, the resolution of these models allows the localization on beta 2 of the antigenic sites recognized by the two antibodies, to show that the C-terminal F2 domains lie at the distal ends of the elongated beta 2 protein, and to show how steric hindrance prevents beta 2, though structurally and functionally dimeric, from binding more than one Fab 93-6 fragment per dimer.     

The binding of lanthanides to non-immune rabbit immunoglobulin G and its fragments.

The binding of Gd(III) to rabbit IgG (immunoglobulin G) and the Fab (N-terminal half of heavy and light chain), (Bab')2 (N-terminal half of heavy and light chains joined by inter-chain disulphide bond), Fc (C-terminal half of heavy-chain dimer)and pFc' (C-terminal quarter of heavy-chain dimer) fragments was demonstrated by measurements of the enhancement of the solvent-water proton relaxation rates in the appropriate Gd(III) solutions. At pH 5.5 there are six specific Gd(III)-binding sites on the IgG. These six sites can be divided into two classes; two very 'tight' sites on the Fc fragment (Kd approx. 5 muM) and two weaker sites on each Fab region (Kd approx. 140 muM). Ca(II) does not apparently compete for these metal-binding sites. The metal-binding parameters for IgG can be explained as the sum of the metal binding to the isolated Fab and Fc fragments, suggesting that there is no apparent interaction between the Fab and Fc regions in the IgG molecule. The binding of Gd(III) to Fab and Fc fragments was also monitored by measuring changes in the electron-spin-resonance spectrum of Gd(III) in the presence of each fragment and also by monitoring the effects of Gd(III) on the protein fluorescence at 340 nm (excitation 295 nm). The fluorescence of Tb(III) solutions of 545 nm (excitation 295 nm) is enhanced slightly on addition of Fab or Fc.     

Investigation of degradation processes in IgG1 monoclonal antibodies by limited proteolysis coupled with weak cation-exchange HPLC

A new cation-exchange high-performance liquid chromatography (HPLC) method that separates fragment antigen-binding (Fab) and fragment crystallizable (Fc) domains generated by the limited proteolysis of monoclonal antibodies (mAbs) was developed. This assay has proven to be suitable for studying complex degradation processes involving various immunoglobulin G1 (IgG1) molecules. Assignment of covalent degradations to specific regions of mAbs was facilitated by using Lys-C and papain to generate Fab and Fc fragments with unique, protease-dependent elution times. In particular, this method was useful for characterizing protein variants formed in the presence of salt under accelerated storage conditions. Two isoforms that accumulated during storage were readily identified as Fab-related species prior to mass-spectrometric analysis. Both showed reduced biological activity likely resulting from modifications within or in proximity of the complementarity-determining regions (CDRs). Utility of this assay was further illustrated in the work to characterize light-induced degradations in mAb formulations. In this case, a previously unknown Fab-related species which populated upon light exposure was observed. This species was well resolved from unmodified Fab, allowing for direct and high-purity fractionation. Mass-spectrometric analysis subsequently identified a histidine-related degradation product associated with the CDR2 of the heavy chain. In addition, the method was applied to assess the structural organization of a noncovalent IgG1 dimer. A new species corresponding to a Fab–Fab complex was found, implying that interactions between Fab domains were responsible for dimerization. Overall, the data presented demonstrate the suitability of this cation-exchange HPLC method for studying a wide range of covalent and noncovalent degradations in IgG1 mAbs.     

Role of antigen and,antibody in the regulation of the immune response

The enzyme dextranase could degrade antigenic dextran in vivo even when given 6-15 d after the antigen. Dextranase injected after the antigen suppressed the immune response when given 24 but not 48 h after the antigen, indicating that the antigen must interact with the immune system for 48 h to initiate a response. Thereafter, the B cells are independent of further antigen stimulation. To show whether antibody-mediated suppression of the immune response was determinant specific FITC-conjugated SRC were applied as immunogen and antibodies were raised both against the carrier (SRC) and the FITC hapten. When these antibodies were injected 1-3 h after the immunogen they only suppressed the immune response to the corresponding determinant. Anti-carrier antibodies usually enhanced the response to the hapten. Therefore, antibody-mediated suppression of the immune response is determinant-specific and cannot be mediated in vivo to a detectable extent by the Fc part of the antibodies.     

Specific recognition of a DNA immunogen by its elicited antibody

DNA recognition by antibodies is a key feature of autoimmune diseases, yet model systems with structural information are very limited. The monoclonal antibody ED-10 recognizes one of the strands of the DNA duplex used in the immunogenic complex. Modifications of the 5' end decrease the binding affinity and short oligonucleotides retain high binding affinity. We determined crystal structures for the Fab bound to a 6-mer oligonucleotide containing the specific sequence that raised the antibody and compared it with the unliganded Fab. Only the first two bases from the 5' end (dTdC) display electron density and we observe four key hydrogen bonds at the interface. The thymine ring is stacked between TrpH50 and TrpH95, and the cytosine ring is packed against TyrL32. Upon DNA binding, TyrH97 and TrpH95 rearrange to allow subnanomolar binding affinity, five orders of magnitude higher than other reported complexes, possibly because of having gone through affinity maturation. This structure represents the first bona fide antibody DNA immunogen complex described in atomic detail.     

Altered dynamics in Lck SH3 upon binding to the LBD1 domain of Herpesvirus saimiri Tip

The Tip protein from Herpesvirus saimiri interacts with the SH3 domain from the Src-family kinase Lck via a proline-containing sequence termed LBD1. Src-family kinase SH3 domains related to Lck have been shown to be dynamic in solution and partially unfold under physiological conditions. The rate of such partial unfolding is reduced by viral protein binding. To determine if the Lck SH3 domain displayed similar behavior, the domain was investigated with hydrogen exchange and mass spectrometry. Lck SH3 was found to be highly dynamic in solution. While other SH3 domains require as much as 10,000 sec to become totally deuterated, Lck SH3 became almost completely labeled within 200 sec. A partial unfolding event involving 8-10 residues was observed with a half-life of approximately 10 sec. Tip LBD1 binding did not cause gross structural changes in Lck SH3 but globally stabilized the domain and reduced the rate of partial unfolding by a factor of five. The region of partial unfolding in Lck SH3 was found to be similar to that identified for other SH3 domains that partially unfold. Although the sequence conservation between Lck SH3 and other closely related SH3 domains is high, the dynamics do not appear to be conserved.     

A highly potent CD73 biparatopic antibody blocks organization of the enzyme active site through dual mechanisms

The dimeric ectonucleotidase CD73 catalyzes the hydrolysis of AMP at the cell surface to form adenosine, a potent suppressor of the immune response. Blocking CD73 activity in the tumor microenvironment can have a beneficial effect on tumor eradication and is a promising approach for cancer therapy. Biparatopic antibodies binding different regions of CD73 may be a means to antagonize its enzymatic activity. A panel of biparatopic antibodies representing the pairwise combination of 11 parental monoclonal antibodies against CD73 was generated by Fab-arm exchange. Nine variants vastly exceeded the potency of their parental antibodies with ≥90% inhibition of activity and subnanomolar EC₅₀ values. Pairing the Fabs of parents with nonoverlapping epitopes was both sufficient and necessary whereas monovalent antibodies were poor inhibitors. Some parental antibodies yielded potent biparatopics with multiple partners, one of which (TB19) producing the most potent. The structure of the TB19 Fab with CD73 reveals that it blocks alignment of the N- and C-terminal CD73 domains necessary for catalysis. A separate structure of CD73 with a Fab (TB38) which complements TB19 in a particularly potent biparatopic shows its binding to a nonoverlapping site on the CD73 N-terminal domain. Structural modeling demonstrates a TB19/TB38 biparatopic antibody would be unable to bind the CD73 dimer in a bivalent manner, implicating crosslinking of separate CD73 dimers in its mechanism of action. This ability of a biparatopic antibody to both crosslink CD73 dimers and fix them in an inactive conformation thus represents a highly effective mechanism for the inhibition of CD73 activity.     

Interaction of human creatine phosphokinase isoenzymes with rabbit antibodies and their Fab-fragments

The interaction of human creatine phosphokinase isoenzymes with rabbit antibodies and their Fab has been studied. It has been shown that Fab of the antibodies against MM or BB isoenzymes preserve high specificity of intact antibodies and the ability to inhibit creatine kinase isoenzymes. Differences between antibodies and their Fab have been found to exist with respect to the kinetics of binding with homologous isoenzymes: the rate of the complex formation for Fab is significantly higher. The interaction of creatine kinase isoenzymes with intact antibodies and their Fab is not affected by the addition of creatine kinase substrates. The antibodies against MM and BB isoenzymes have been used to study the individual properties of each subunit of the M- and B-type in a hybrid dimer MB. It has been shown that such properties of these subunits as the Michaelis constants, pH dependence and inhibition by homologous antibodies are identical to those of non-hybrid MM and BB isoenzymes, respectively.     

Hydrogen exchange shows peptide binding stabilizes motions in Hck SH2.

Src-homology-2 domains are small, 100 amino acid protein modules that are present in a number of signal transduction proteins. Previous NMR studies of SH2 domain dynamics indicate that peptide binding decreases protein motions in the pico- to nanosecond, and perhaps slower, time range. We suggest that amide hydrogen exchange and mass spectrometry may be useful for detecting changes in protein dynamics because hydrogen exchange rates are relatively insensitive to the time domains of the dynamics. In the present study, hydrogen exchange and mass spectrometry were used to probe hematopoietic cell kinase SH2 that was either free or bound to a 12-residue high-affinity peptide. Hydrogen exchange rates were determined by exposing free and bound SH2 to D(2)O, fragmenting the SH2 with pepsin, and determining the deuterium level in the peptic fragments. Binding generally decreased hydrogen exchange along much of the SH2 backbone, indicating a widespread reduction in dynamics. Alterations in the exchange of the most rapidly exchanging amide hydrogens, which was detected following acid quench and analysis by mass spectrometry, were used to locate differences in low-amplitude motion when SH2 was bound to the peptide. In addition, the results indicate that hydrogen exchange from the folded form of SH2 is an important process along the entire SH2 backbone.     

Characterization of the interface structure of enzyme-inhibitor complex by using hydrogen-deuterium exchange and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

We investigated the interaction between a thiol protease inhibitor, cystatin, and its target enzyme, papain, by hydrogen-deuterium (H/D) exchange in conjunction with successive analysis by collision-induced dissociation (CID) in an rf-only hexapole ion guide with electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS). The deuterium incorporation into backbone amide hydrogens of cystatin was analyzed at different time points in the presence or absence of papain, examining the mass of each fragment produced by hexapole-CID. In the absence of papain, amide hydrogens in short amino-terminal fragments, such as b10(2+) and b12(2+), were highly deuterated within 1 min. Although fewer fragments were observed for the cystatin-papain complex in the hexapole-CID spectra, significant reductions in initial deuterium content were recognized throughout the sequence of cystatin. This suggests that complex formation restricted the flexibility of the whole cystatin molecule. Detailed analyses revealed that a marked reduction in deuterium content in the region of residues 1-10 persisted for hours, suggesting that the flexible N-terminal region was tightly fixed in the binding pocket with hydrogen bonds. Our results are consistent with those of previous studies on the structure and inhibition mechanism of cystatin. We demonstrated here that enzyme-inhibitor interactions can be characterized by H/D exchange in combination with CID in a hexapole ion guide using ESI-FTICR MS rapidly and using only a small amount of sample.     

Complexes of Neutralizing and Non-Neutralizing Affinity Matured Fabs with a Mimetic of the Internal Trimeric Coiled-Coil of HIV-1 gp41

A series of mini-antibodies (monovalent and bivalent Fabs) targeting the conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 has been previously constructed and reported. Crystal structures of two closely related monovalent Fabs, one (Fab 8066) broadly neutralizing across a wide panel of HIV-1 subtype B and C viruses, and the other (Fab 8062) non-neutralizing, representing the extremes of this series, were previously solved as complexes with 5-Helix, a gp41 pre-hairpin intermediate mimetic. Binding of these Fabs to covalently stabilized chimeric trimers of N-peptides of HIV-1 gp41 (named (CCIZN36)₃ or 3-H) has now been investigated using X-ray crystallography, cryo-electron microscopy, and a variety of biophysical methods. Crystal structures of the complexes between 3-H and Fab 8066 and Fab 8062 were determined at 2.8 and 3.0 Å resolution, respectively. Although the structures of the complexes with the neutralizing Fab 8066 and its non-neutralizing counterpart Fab 8062 were generally similar, small differences between them could be correlated with the biological properties of these antibodies. The conformations of the corresponding CDRs of each antibody in the complexes with 3-H and 5-Helix are very similar. The adaptation to a different target upon complex formation is predominantly achieved by changes in the structure of the trimer of N-HR helices, as well as by adjustment of the orientation of the Fab molecule relative to the N-HR in the complex, via rigid-body movement. The structural data presented here indicate that binding of three Fabs 8062 with high affinity requires more significant changes in the structure of the N-HR trimer compared to binding of Fab 8066. A comparative analysis of the structures of Fabs complexed to different gp41 intermediate mimetics allows further evaluation of biological relevance for generation of neutralizing antibodies, as well as provides novel structural insights into immunogen design.     

Antibody against the amino terminus of alpha-actin inhibits actomyosin interactions in the presence of ATP

Actomyosin interactions in the presence of ATP were examined by using site-specific antibodies directed against the first seven N-terminal residues on skeletal alpha-actin. Fab fragments of these antibodies (S alpha N Fab) inhibited effectively the actin-activated ATPase of myosin subfragment 1 (S-1) at both 5 and 25 degrees C. Binding experiments carried out in the presence of ATP at 5 degrees C revealed that the catalytic inhibition was related to the inhibition of S-1 binding to actin by Fab. At equimolar ratios of Fab to actin, the binding of S-1 to actin and the activated ATPase were inhibited by 75 and 82%, respectively. These results, when contrasted with the small effect of Fab on rigor actomyosin binding, suggest ATP-induced changes at the interface of actin and myosin.     

Hydrogen-deuterium exchange in gamma-irradiated polycrystalline DL-alanine: a spin-trapping and e.s.r. study

The hydrogen-deuterium exchange reactions in gamma-irradiated DL-alanine in the solid state were investigated by spin-trapping and electron spin resonance (e.s.r) using selectively deuterated DL-alanine. Subsequent to gamma-radiolysis at 30 degrees C, polycrystalline DL-alanine was dissolved in aqueous solutions of 2-methyl-2-nitrosopropane and the extent of H-D exchange of the deamination radicals was followed by e.s.r. After formation of the deamination radicals, four exchange reactions were found to occur between the radicals and the surrounding undamaged molecules. The first reaction, which occurs between the hydrogens of the C-2 carbon of the radicals and those of the methyl groups of the neighbouring molecules, can be followed at room temperature. The three other reactions could be conveniently monitored in gamma-irradiated polycrystalline alanine at 110 degrees C. The first of the other three reactions takes place between the methyl hydrogens of the radicals and the C-2 hydrogens of nearby molecules, while the remaining processes involve exchange between the hydrogen atoms of the amino group and those on the C-2 and C-3 carbon atoms of the deamination radical.     

2G12-Expressing B Cell Lines May Aid in HIV Carbohydrate Vaccine Design Strategies

The highly conserved cluster of high-mannose glycans on the HIV-1 envelope glycoprotein, gp120, has been highlighted as a target for neutralizing antibodies. 2G12, the first HIV-1 antiglycan neutralizing antibody described, binds with an unusual domain-exchanged structure that creates a high-affinity multivalent binding surface. It is an interesting challenge for rational vaccine design to generate immunogens capable of eliciting domain-exchanged 2G12-like responses. We recently showed that di-mannose recognition by the variable domains of 2G12 is independent of domain exchange but that exchange is critical for virus neutralization. Carbohydrate-based immunogens aimed at inducing 2G12-like antibodies may need to drive both di-mannose recognition and domain exchange through interactions with B cell receptors. Here we assessed the ability of such immunogens to activate mouse B cell lines displaying domain-exchanged wild-type 2G12 (2G12 WT), a non-domain-exchanged Y-shaped variant (2G12 I19R), and germ line 2G12 (2G12 gl). We show that several immunogens, including heat-killed yeast and bacteria, can activate both 2G12 WT and 2G12 I19R B cells. However, only discrete clusters of high-mannose glycans, as on recombinant forms of the HIV-1 envelope trimer and oligodendrons, activate 2G12 WT B cells. Furthermore, no immunogen tested activated 2G12 gl cells. Our results support the hypothesis that in order to drive domain exchange of an antimannose antibody response, a boost with an immunogen displaying discrete clusters of high-mannose glycans not recognized by conventional Y-shaped antibodies will be required. Additionally, a molecule capable of activating 2G12 gl cells might also be required. The results highlight broadly neutralizing antibody-expressing mouse B cells as potentially useful tools for carbohydrate immunogen screening.     

Structure of the discoidin domain receptor 1 extracellular region bound to an inhibitory Fab fragment reveals features important for signaling

The discoidin domain receptors, DDR1 and DDR2, are constitutively dimeric receptor tyrosine kinases that are activated by triple-helical collagen. Aberrant DDR signaling contributes to several human pathologies, including many cancers. We have generated monoclonal antibodies (mAbs) that inhibit DDR1 signaling without interfering with collagen binding. The crystal structure of the monomeric DDR1 extracellular region bound to the Fab fragment of mAb 3E3 reveals that the collagen-binding discoidin (DS) domain is tightly associated with the following DS-like domain, which contains the epitopes of all mAbs. A conserved surface patch in the DS domain outside the collagen-binding site is shown to be required for signaling. Thus, the active conformation of the DDR1 dimer involves collagen-induced contacts between the DS domains, in addition to the previously identified association of transmembrane helices. The mAbs likely inhibit signaling by sterically blocking the extracellular association of DDR1 subunits.     

Dissecting interdomain communication within cAPK regulatory subunit type IIbeta using enhanced amide hydrogen/deuterium exchange mass spectrometry (DXMS)

cAMP-dependent protein kinase (cAPK) is a heterotetramer containing a regulatory (R) subunit dimer bound to two catalytic (C) subunits and is involved in numerous cell signaling pathways. The C-subunit is activated allosterically when two cAMP molecules bind sequentially to the cAMP-binding domains, designated A and B (cAB-A and cAB-B, respectively). Each cAMP-binding domain contains a conserved Arg residue that is critical for high-affinity cAMP binding. Replacement of this Arg with Lys affects cAMP affinity, the structural integrity of the cAMP-binding domains, and cAPK activation. To better understand the local and long-range effects that the Arg-to-Lys mutation has on the dynamic properties of the R-subunit, the amide hydrogen/deuterium exchange in the RIIbeta subunit was probed by electrospray mass spectrometry. Mutant proteins containing the Arg-to-Lys substitution in either cAMP-binding domain were deuterated for various times and then, prior to mass spectrometry analysis, subjected to pepsin digestion to localize the deuterium incorporation. Mutation of this Arg in cAB-A (Arg230) causes an increase in amide hydrogen exchange throughout the mutated domain that is beyond the modest and localized effects of cAMP removal and is indicative of the importance of this Arg in domain organization. Mutation of Arg359 (cAB-B) leads to increased exchange in the adjacent cAB-A domain, particularly in the cAB-A domain C-helix that lies on top of the cAB-B domain and is believed to be functionally linked to the cAB-B domain. This interdomain communication appears to be a unidirectional pathway, as mutation of Arg230 in cAB-A does not effect dynamics of the cAB-B domain.     

Characterization of transient protein folding intermediates during myoglobin reconstitution by time-resolved electrospray mass spectrometry with on-line isotopic pulse labeling

A novel technique for studying protein folding kinetics is presented. It is based on a continuous-flow setup that is coupled to an electrospray (ESI) mass spectrometer and allows initiation of a folding reaction, followed by isotopic pulse labeling. The protein is electrosprayed "quasi-instantaneously" after exposure to the deuterated solvent. This approach yields structural information from the ESI charge state distribution and from the H/D exchange levels of individual protein states, while at the same time noncovalent interactions can be monitored. This technique is used to study the reconstitution of holomyoglobin (hMb) from unfolded apomyoglobin (aMb) and free heme. MS/MS is used to establish that a short-lived folding intermediate with two heme groups attached represents a protein-bound heme dimer. This state appears to have a compactness close to that of native hMb; however, isotopic labeling indicates a significantly perturbed structure. Another intermediate is bound to a single heme group and shows a charge state distribution similar to that of unfolded aMb. Exchange levels exhibited by this state are lower than for unfolded aMb, indicating that fewer hydrogens are exposed to the solvent and/or that more of them are involved in hydrogen bonding. Native hMb leads to the formation of low charge state ions (hMb(9+), hMb(8+)) and shows low exchange levels. However, early during reconstitution, a slightly unfolded form of the heme-protein complex contributes to the observed hMb(9+) ions. A peak width analysis reveals that the structural heterogeneity of some of the observed protein species decreases as reconstitution proceeds.     

An inhibitory monoclonal antibody binds at the turn of the helix-turn-helix motif in the N-terminal domain of HIV-1 integrase

With the increase in our understanding of its structure and enzymatic mechanism, HIV-1 integrase (IN) has become a promising target for designing drugs to treat patients with AIDS. To investigate the structure and function of IN, a panel of monoclonal antibodies (mAbs) directed against HIV-1 IN was raised and characterized previously in this laboratory. Among them, mAbs17, -4, and -33 were found to inhibit IN activity in vitro. In this study, we investigated the interaction of N-terminal-specific mAb17 and its isolated Fab fragment with full-length HIV-1 IN(1-288) and its isolated N-terminal, Zn(2+)-binding domain IN(1-49). Our results show that binding of Zn(2+) to IN(1-49) stabilizes the mAb17-IN complex and that dimer dissociation is not required for binding of the Fab. To identify the epitope recognized by mAb17, we developed a protein footprinting technique based on controlled proteolysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Binding was mapped to a region within amino acids Asp(25)-Glu(35). This peptide corresponds to the end of a helix-turn-helix motif in the IN(1-55) NMR structure and contributes to the dimerization of the N-terminal domain. Antibody binding also appears to destabilize the N-terminal helix in this domain. A molecular model of the [IN(1-49)](2).(Fab)(1) complex shows Fab binding across the dimer protein and suggests a potential target for drug design. These data also suggest that mAb17 inhibits integrase activity by blocking critical protein-protein interactions and/or by distorting the orientation of the N-terminal alpha-helix. The relevance of our results to an understanding of IN function is discussed.