Profiling the Biophysical Developability Properties of Common IgG1 Fc Effector Silencing Variants.

Therapeutic antibodies represent the most significant modality in biologics, with around 150 approved drugs on the market. In addition to specific target binding mediated by the variable fragments (Fvs) of the heavy and light chains, antibodies possess effector functions through binding of the constant region (Fc) to Fcγ receptors (FcγR), which allow immune cells to attack and kill target cells using a variety of mechanisms. However, for some applications, including T-cell-engaging bispecifics, this effector function is typically undesired. Mutations within the lower hinge and the second constant domain (CH2) of IgG1 that comprise the FcγR binding interface reduce or eliminate effector function ("Fc silencing") while retaining binding to the neonatal Fc receptor (FcRn), important for normal antibody pharmacokinetics (PKs). Comprehensive profiling of biophysical developability properties would benefit the choice of constant region variants for development. Here, we produce a large panel of representative mutations previously described in the literature and in many cases in clinical or approved molecules, generate select combinations thereof, and characterize their binding and biophysical properties. We find that some commonly used CH2 mutations, including D265A and P331S, are effective in reducing binding to FcγR but significantly reduce stability, promoting aggregation, particularly under acidic conditions commonly employed in manufacturing. We highlight mutation sets that are particularly effective for eliminating Fc effector function with the retention of WT-like stability, including L234A, L235A, and S267K (LALA-S267K), L234A, L235E, and S267K (LALE-S267K), L234A, L235A, and P329A (LALA-P329A), and L234A, L235E, and P329G (LALE-P329G).

Structure-based engineering of a novel CD3ε-targeting antibody for reduced polyreactivity.

Bispecific antibodies continue to represent a growth area for antibody therapeutics, with roughly a third of molecules in clinical development being T-cell engagers that use an anti-CD3 binding arm. CD3 antibodies possessing cross-reactivity with cynomolgus monkey typically recognize a highly electronegative linear epitope at the extreme N-terminus of CD3 epsilon (CD3ε). Such antibodies have high isoelectric points and display problematic polyreactivity (correlated with poor pharmacokinetics for monospecific antibodies). Using insights from the crystal structure of anti-Hu/Cy CD3 antibody ADI-26906 in complex with CD3ε and antibody engineering using a yeast-based platform, we have derived high-affinity CD3 antibody variants with very low polyreactivity and significantly improved biophysical developability. Comparison of these variants with CD3 antibodies in the clinic (as part of bi- or multi-specifics) shows that affinity for CD3 is correlated with polyreactivity. Our engineered CD3 antibodies break this correlation, forming a broad affinity range with no to low polyreactivity. Such antibodies will enable bispecifics with improved pharmacokinetic and safety profiles and suggest engineering solutions that will benefit the large and growing sector of T-cell engagers.

Influences on trimerization and aggregation of soluble, cleaved HIV-1 SOSIP envelope glycoprotein.

We describe methods to improve the properties of soluble, cleaved gp140 trimers of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) for use in structural studies and as immunogens. In the absence of nonionic detergents, gp140 of the KNH1144 genotype, terminating at residue 681 in gp41 (SOSIP.681), has a tendency to form higher-order complexes or aggregates, which is particularly undesirable for structure-based research. We found that this aggregation in the absence of detergent does not involve the V1, V2, or V3 variable regions of gp120. Moreover, we observed that detergent forms micelles around the membrane-proximal external region (MPER) of the SOSIP.681 gp140 trimers, whereas deletion of most of the MPER residues by terminating the gp140 at residue 664 (SOSIP.664) prevented the aggregation that otherwise occurs in SOSIP.681 in the absence of detergent. Although the MPER can contribute to trimer formation, truncation of most of it only modestly reduced trimerization and lacked global adverse effects on antigenicity. Thus, the MPER deletion minimally influenced the kinetics of the binding of soluble CD4 and a CD4-binding site antibody to immobilized trimers, as detected by surface plasmon resonance. Furthermore, the MPER deletion did not alter the overall three-dimensional structure of the trimers, as viewed by negative-stain electron microscopy. Homogeneous and aggregate-free MPER-truncated SOSIP Env trimers are therefore useful for immunogenicity and structural studies.

Broadly neutralizing antibody PGT121 allosterically modulates CD4 binding via recognition of the HIV-1 gp120 V3 base and multiple surrounding glycans.

New broad and potent neutralizing HIV-1 antibodies have recently been described that are largely dependent on the gp120 N332 glycan for Env recognition. Members of the PGT121 family of antibodies, isolated from an African donor, neutralize ∼70% of circulating isolates with a median IC50 less than 0.05 µg ml(-1). Here, we show that three family members, PGT121, PGT122 and PGT123, have very similar crystal structures. A long 24-residue HCDR3 divides the antibody binding site into two functional surfaces, consisting of an open face, formed by the heavy chain CDRs, and an elongated face, formed by LCDR1, LCDR3 and the tip of the HCDR3. Alanine scanning mutagenesis of the antibody paratope reveals a crucial role in neutralization for residues on the elongated face, whereas the open face, which accommodates a complex biantennary glycan in the PGT121 structure, appears to play a more secondary role. Negative-stain EM reconstructions of an engineered recombinant Env gp140 trimer (SOSIP.664) reveal that PGT122 interacts with the gp120 outer domain at a more vertical angle with respect to the top surface of the spike than the previously characterized antibody PGT128, which is also dependent on the N332 glycan. We then used ITC and FACS to demonstrate that the PGT121 antibodies inhibit CD4 binding to gp120 despite the epitope being distal from the CD4 binding site. Together, these structural, functional and biophysical results suggest that the PGT121 antibodies may interfere with Env receptor engagement by an allosteric mechanism in which key structural elements, such as the V3 base, the N332 oligomannose glycan and surrounding glycans, including a putative V1/V2 complex biantennary glycan, are conformationally constrained.

Partial enzymatic deglycosylation preserves the structure of cleaved recombinant HIV-1 envelope glycoprotein trimers.

The trimeric envelope glycoprotein complex (Env) is the focus of vaccine development programs aimed at generating protective humoral responses to human immunodeficiency virus type 1 (HIV-1). N-Linked glycans, which constitute almost half of the molecular mass of the external Env domains, produce considerable structural heterogeneity and are a major impediment to crystallization studies. Moreover, by shielding the peptide backbone, glycans can block attempts to generate neutralizing antibodies against a substantial subset of potential epitopes when Env proteins are used as immunogens. Here, we describe the partial deglycosylation of soluble, cleaved recombinant Env trimers by inhibition of the synthesis of complex N-glycans during Env production, followed by treatment with glycosidases under conditions that preserve Env trimer integrity. The partially deglycosylated trimers are stable, and neither abnormally sensitive to proteolytic digestion nor prone to aggregation. Moreover, the deglycosylated trimers retain or increase their ability to bind CD4 and antibodies that are directed to conformational epitopes, including the CD4-binding site and the V3 region. However, as expected, they do not react with glycan-dependent antibodies 2G12 and PGT123, or the C-type lectin receptor DC-SIGN. Electron microscopic analysis shows that partially deglycosylated trimers have a structure similar to fully glycosylated trimers, indicating that removal of glycans does not substantially perturb the structural integrity of the trimer. The glycan-depleted Env trimers should be useful for structural and immunogenicity studies.

Structure of HIV-1 gp120 V1/V2 domain with broadly neutralizing antibody PG9.

Variable regions 1 and 2 (V1/V2) of human immunodeficiency virus-1 (HIV-1) gp120 envelope glycoprotein are critical for viral evasion of antibody neutralization, and are themselves protected by extraordinary sequence diversity and N-linked glycosylation. Human antibodies such as PG9 nonetheless engage V1/V2 and neutralize 80% of HIV-1 isolates. Here we report the structure of V1/V2 in complex with PG9. V1/V2 forms a four-stranded ß-sheet domain, in which sequence diversity and glycosylation are largely segregated to strand-connecting loops. PG9 recognition involves electrostatic, sequence-independent and glycan interactions: the latter account for over half the interactive surface but are of sufficiently weak affinity to avoid autoreactivity. The structures of V1/V2-directed antibodies CH04 and PGT145 indicate that they share a common mode of glycan penetration by extended anionic loops. In addition to structurally defining V1/V2, the results thus identify a paradigm of antibody recognition for highly glycosylated antigens, which-with PG9-involves a site of vulnerability comprising just two glycans and a strand.

Rapid development of glycan-specific, broad, and potent anti-HIV-1 gp120 neutralizing antibodies in an R5 SIV/HIV chimeric virus infected macaque.

It is widely believed that the induction of a broadly neutralizing antibody (bNAb) response will be a critical component of a successful vaccine against HIV. A significant fraction of HIV-infected individuals mount bNAb responses, providing support for the notion that such responses could be elicited through vaccination. Infection of macaques with simian immunodeficiency virus (SIV) or SIV/HIV chimeric virus (SHIV) has been widely used to model aspects of HIV infection, but to date, only limited bNAb responses have been described. Here, we screened plasma from 14 R5-tropic SHIV-infected macaques for broadly neutralizing activity and identified a macaque with highly potent cross-clade plasma NAb response. Longitudinal studies showed that the development of broad and autologous NAb responses occurred coincidentally in this animal. Serum-mapping studies, using pseudovirus point mutants and antigen adsorption assays, indicated that the plasma bNAbs are specific for epitopes that include carbohydrates and are critically dependent on the glycan at position 332 of Env gp120. The results described herein provide insight into the development and evolution of a broad response, suggest that certain bNAb specificities may be more rapidly induced by immunization than others, and provide a potential model for the facile study of the development of bNAb responses.

A potent and broad neutralizing antibody recognizes and penetrates the HIV glycan shield.

The HIV envelope (Env) protein gp120 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to interact directly with the HIV glycan coat. Crystal structures of antigen-binding fragments (Fabs) PGT 127 and 128 with Man(9) at 1.65 and 1.29 angstrom resolution, respectively, and glycan binding data delineate a specific high mannose-binding site. Fab PGT 128 complexed with a fully glycosylated gp120 outer domain at 3.25 angstroms reveals that the antibody penetrates the glycan shield and recognizes two conserved glycans as well as a short ß-strand segment of the gp120 V3 loop, accounting for its high binding affinity and broad specificity. Furthermore, our data suggest that the high neutralization potency of PGT 127 and 128 immunoglobulin Gs may be mediated by cross-linking Env trimers on the viral surface.

Structure-based design of a protein immunogen that displays an HIV-1 gp41 neutralizing epitope.

Antibody Z13e1 is a relatively broadly neutralizing anti-human immunodeficiency virus type 1 antibody that recognizes the membrane-proximal external region (MPER) of the human immunodeficiency virus type 1 envelope glycoprotein gp41. Based on the crystal structure of an MPER epitope peptide in complex with Z13e1 Fab, we identified an unrelated protein, interleukin (IL)-22, with a surface-exposed region that is structurally homologous in its backbone to the gp41 Z13e1 epitope. By grafting the gp41 Z13e1 epitope sequence onto the structurally homologous region in IL-22, we engineered a novel protein (Z13-IL22-2) that contains the MPER epitope sequence for use as a potential immunogen and as a reagent for the detection of Z13e1-like antibodies. The Z13-IL22-2 protein binds Fab Z13e1 with a K(d) of 73 nM. The crystal structure of Z13-IL22-2 in complex with Fab Z13e1 shows that the epitope region is faithfully replicated in the Fab-bound scaffold protein; however, isothermal calorimetry studies indicate that Fab binding to Z13-IL22-2 is not a lock-and-key event, leaving open the question of whether conformational changes upon binding occur in the Fab, in Z13-IL-22, or in both.

Broad neutralization coverage of HIV by multiple highly potent antibodies.

Broadly neutralizing antibodies against highly variable viral pathogens are much sought after to treat or protect against global circulating viruses. Here we probed the neutralizing antibody repertoires of four human immunodeficiency virus (HIV)-infected donors with remarkably broad and potent neutralizing responses and rescued 17 new monoclonal antibodies that neutralize broadly across clades. Many of the new monoclonal antibodies are almost tenfold more potent than the recently described PG9, PG16 and VRC01 broadly neutralizing monoclonal antibodies and 100-fold more potent than the original prototype HIV broadly neutralizing monoclonal antibodies. The monoclonal antibodies largely recapitulate the neutralization breadth found in the corresponding donor serum and many recognize novel epitopes on envelope (Env) glycoprotein gp120, illuminating new targets for vaccine design. Analysis of neutralization by the full complement of anti-HIV broadly neutralizing monoclonal antibodies now available reveals that certain combinations of antibodies should offer markedly more favourable coverage of the enormous diversity of global circulating viruses than others and these combinations might be sought in active or passive immunization regimes. Overall, the isolation of multiple HIV broadly neutralizing monoclonal antibodies from several donors that, in aggregate, provide broad coverage at low concentrations is a highly positive indicator for the eventual design of an effective antibody-based HIV vaccine.

Trimeric HIV-1 glycoprotein gp140 immunogens and native HIV-1 envelope glycoproteins display the same closed and open quaternary molecular architectures.

The initial step in HIV-1 infection occurs with the binding of cell surface CD4 to trimeric HIV-1 envelope glycoproteins (Env), a heterodimer of a transmembrane glycoprotein (gp41) and a surface glycoprotein (gp120). The design of soluble versions of trimeric Env that display structural and functional properties similar to those observed on intact viruses is highly desirable from the viewpoint of designing immunogens that could be effective as vaccines against HIV/AIDS. Using cryoelectron tomography combined with subvolume averaging, we have analyzed the structure of SOSIP gp140 trimers, which are cleaved, solubilized versions of the ectodomain of trimeric HIV-1 Env. We show that unliganded gp140 trimers adopt a quaternary arrangement similar to that displayed by native unliganded trimers on the surface of intact HIV-1 virions. When complexed with soluble CD4, Fab 17b, which binds to gp120 at its chemokine coreceptor binding site, or both soluble CD4 and 17b Fab, gp140 trimers display an open conformation in which there is an outward rotation and displacement of each gp120 protomer. We demonstrate that the molecular arrangements of gp120 trimers in the closed and open conformations of the soluble trimer are the same as those observed for the closed and open states, respectively, of trimeric gp120 on intact HIV-1 BaL virions, establishing that soluble gp140 trimers can be designed to mimic the quaternary structural transitions displayed by native trimeric Env.

Structure-based vaccine design in HIV: blind men and the elephant?

Traditional vaccine approaches have failed for HIV and novel strategies are now being sought to develop immunogens designed to elicit specific activity against known broad neutralization epitopes. Structure-based vaccine design has great potential but, thus far, remains a largely unproven concept. Further structural information for the envelope (Env) glycoproteins, gp120 and gp41, is needed, particularly for understanding trimer-specific antibodies and their epitopes and to clarify atomic details of the structural elements responsible for masking crucial epitopes and for mediating the conformational rearrangements undertaken during the process of receptor-binding and membrane fusion.

Structure and function of broadly reactive antibody PG16 reveal an H3 subdomain that mediates potent neutralization of HIV-1.

Development of an effective vaccine against HIV-1 will likely require elicitation of broad and potent neutralizing antibodies against the trimeric surface envelope glycoprotein (Env). Monoclonal antibodies (mAbs) PG9 and PG16 neutralize approximately 80% of HIV-1 isolates across all clades with extraordinary potency and target novel epitopes preferentially expressed on Env trimers. As these neutralization properties are ideal for a vaccine-elicited antibody response to HIV-1, their structural basis was investigated. The crystal structure of the antigen-binding fragment (Fab) of PG16 at 2.5 A resolution revealed its unusually long, 28-residue, complementarity determining region (CDR) H3 forms a unique, stable subdomain that towers above the antibody surface. A 7-residue "specificity loop" on the "hammerhead" subdomain was identified that, when transplanted from PG16 to PG9 and vice versa, accounted for differences in the fine specificity and neutralization of these two mAbs. The PG16 electron density maps also revealed that a CDR H3 tyrosine was sulfated, which was confirmed for both PG9 (doubly) and PG16 (singly) by mass spectral analysis. We further showed that tyrosine sulfation plays a role in binding and neutralization. An N-linked glycan modification is observed in the variable light chain, but not required for antigen recognition. Further, the crystal structure of the PG9 light chain at 3.0 A facilitated homology modeling to support the presence of these unusual features in PG9. Thus, PG9 and PG16 use unique structural features to mediate potent neutralization of HIV-1 that may be of utility in antibody engineering and for high-affinity recognition of a variety of therapeutic targets.

A conformational switch in human immunodeficiency virus gp41 revealed by the structures of overlapping epitopes recognized by neutralizing antibodies.

The membrane-proximal external region (MPER) of the human immunodeficiency virus (HIV) envelope glycoprotein (gp41) is critical for viral fusion and infectivity and is the target of three of the five known broadly neutralizing HIV type 1 (HIV-1) antibodies, 2F5, Z13, and 4E10. Here, we report the crystal structure of the Fab fragment of Z13e1, an affinity-enhanced variant of monoclonal antibody Z13, in complex with a 12-residue peptide corresponding to the core epitope (W(670)NWFDITN(677)) at 1.8-A resolution. The bound peptide adopts an S-shaped conformation composed of two tandem, perpendicular helical turns. This conformation differs strikingly from the alpha-helical structure adopted by an overlapping MPER peptide bound to 4E10. Z13e1 binds to an elbow in the MPER at the membrane interface, making relatively few interactions with conserved aromatics (Trp672 and Phe673) that are critical for 4E10 recognition. The comparison of the Z13e1 and 4E10 epitope structures reveals a conformational switch such that neutralization can occur by the recognition of the different conformations and faces of the largely amphipathic MPER. The Z13e1 structure provides significant new insights into the dynamic nature of the MPER, which likely is critical for membrane fusion, and it has significant implications for mechanisms of HIV-1 neutralization by MPER antibodies and for the design of HIV-1 immunogens.

Metal active site elasticity linked to activation of homocysteine in methionine synthases.

Enzymes possessing catalytic zinc centers perform a variety of fundamental processes in nature, including methyl transfer to thiols. Cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthases are two such enzyme families. Although they perform the same net reaction, transfer of a methyl group from methyltetrahydrofolate to homocysteine (Hcy) to form methionine, they display markedly different catalytic strategies, modular organization, and active site zinc centers. Here we report crystal structures of zinc-replete MetE and MetH, both in the presence and absence of Hcy. Structural investigation of the catalytic zinc sites of these two methyltransferases reveals an unexpected inversion of zinc geometry upon binding of Hcy and displacement of an endogenous ligand in both enzymes. In both cases a significant movement of the zinc relative to the protein scaffold accompanies inversion. These structures provide new information on the activation of thiols by zinc-containing enzymes and have led us to propose a paradigm for the mechanism of action of the catalytic zinc sites in these and related methyltransferases. Specifically, zinc is mobile in the active sites of MetE and MetH, and its dynamic nature helps facilitate the active site conformational changes necessary for thiol activation and methyl transfer.

Structural perturbations in the Ala --> Val polymorphism of methylenetetrahydrofolate reductase: how binding of folates may protect against inactivation.

In human methylenetetrahydrofolate reductase (MTHFR) the Ala222Val (677C-->T) polymorphism encodes a heat-labile gene product that is associated with elevated levels of homocysteine and possibly with risk for cardiovascular disease. Generation of the equivalent Ala to Val mutation in Escherichia coli MTHFR, which is 30% identical to the catalytic domain of the human enzyme, creates a protein with enhanced thermolability. In both human and E. coli MTHFR, the A --> V mutation increases the rate of dissociation of FAD, and in both enzymes, loss of FAD is linked to changes in quaternary structure [Yamada, K., Chen, Z., Rozen, R., and Matthews, R. G. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 14853-14858; Guenther, B. D., Sheppard, C. A., Tran, P., Rozen, R., Matthews, R. G., and Ludwig, M. L. (1999) Nat. Struct. Biol. 6, 359-365]. Folates have been shown to protect both human and bacterial enzymes from loss of FAD. Despite its effect on affinity for FAD, the A --> V mutation is located at the bottom of the (betaalpha)(8) barrel of the catalytic domain in a position that does not contact the bound FAD prosthetic group. Here we report the structures of the Ala177Val mutant of E. coli MTHFR and of its complex with the 5,10-dideazafolate analogue, LY309887, and suggest mechanisms by which the mutation may perturb FAD binding. Helix alpha5, which immediately precedes the loop bearing the mutation, carries several residues that interact with FAD, including Asn168, Arg171, and Lys172. In the structures of the mutant enzyme this helix is displaced, perturbing protein-FAD interactions. In the complex with LY309887, the pterin-like ring of the analogue stacks against the si face of the flavin and is secured by hydrogen bonds to residues Gln183 and Asp120 that adjoin this face. The direct interactions of bound folate with the cofactor provide one mechanism for linkage between binding of FAD and folate binding that could account in part for the protective action of folates. Conformation changes induced by folate binding may also suppress dissociation of FAD.

Structures of NADH and CH3-H4folate complexes of Escherichia coli methylenetetrahydrofolate reductase reveal a spartan strategy for a ping-pong reaction.

Methylenetetrahydrofolate reductases (MTHFRs; EC 1.7.99.5) catalyze the NAD(P)H-dependent reduction of 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate) to 5-methyltetrahydrofolate (CH(3)-H(4)folate) using flavin adenine dinucleotide (FAD) as a cofactor. The initial X-ray structure of Escherichia coli MTHFR revealed that this 33-kDa polypeptide is a (betaalpha)(8) barrel that aggregates to form an unusual tetramer with only 2-fold symmetry. Structures of reduced enzyme complexed with NADH and of oxidized Glu28Gln enzyme complexed with CH(3)-H(4)folate have now been determined at resolutions of 1.95 and 1.85 A, respectively. The NADH complex reveals a rare mode of dinucleotide binding; NADH adopts a hairpin conformation and is sandwiched between a conserved phenylalanine, Phe223, and the isoalloxazine ring of FAD. The nicotinamide of the bound pyridine nucleotide is stacked against the si face of the flavin ring with C4 adjoining the N5 of FAD, implying that this structure models a complex that is competent for hydride transfer. In the complex with CH(3)-H(4)folate, the pterin ring is also stacked against FAD in an orientation that is favorable for hydride transfer. Thus, the binding sites for the two substrates overlap, as expected for many enzymes that catalyze ping-pong reactions, and several invariant residues interact with both folate and pyridine nucleotide substrates. Comparisons of liganded and substrate-free structures reveal multiple conformations for the loops beta2-alpha2 (L2), beta3-alpha3 (L3), and beta4-alpha4 (L4) and suggest that motions of these loops facilitate the ping-pong reaction. In particular, the L4 loop adopts a "closed" conformation that allows Asp120 to hydrogen bond to the pterin ring in the folate complex but must move to an "open" conformation to allow NADH to bind.

Cobalamin-independent methionine synthase (MetE): a face-to-face double barrel that evolved by gene duplication.

Cobalamin-independent methionine synthase (MetE) catalyzes the transfer of a methyl group from methyltetrahydrofolate to L-homocysteine (Hcy) without using an intermediate methyl carrier. Although MetE displays no detectable sequence homology with cobalamin-dependent methionine synthase (MetH), both enzymes require zinc for activation and binding of Hcy. Crystallographic analyses of MetE from T. maritima reveal an unusual dual-barrel structure in which the active site lies between the tops of the two (betaalpha)(8) barrels. The fold of the N-terminal barrel confirms that it has evolved from the C-terminal polypeptide by gene duplication; comparisons of the barrels provide an intriguing example of homologous domain evolution in which binding sites are obliterated. The C-terminal barrel incorporates the zinc ion that binds and activates Hcy. The zinc-binding site in MetE is distinguished from the (Cys)(3)Zn site in the related enzymes, MetH and betaine-homocysteine methyltransferase, by its position in the barrel and by the metal ligands, which are histidine, cysteine, glutamate, and cysteine in the resting form of MetE. Hcy associates at the face of the metal opposite glutamate, which moves away from the zinc in the binary E.Hcy complex. The folate substrate is not intimately associated with the N-terminal barrel; instead, elements from both barrels contribute binding determinants in a binary complex in which the folate substrate is incorrectly oriented for methyl transfer. Atypical locations of the Hcy and folate sites in the C-terminal barrel presumably permit direct interaction of the substrates in a ternary complex. Structures of the binary substrate complexes imply that rearrangement of folate, perhaps accompanied by domain rearrangement, must occur before formation of a ternary complex that is competent for methyl transfer.

Comparison of functional domains in vertebrate-type ferredoxins.

The vertebrate-type Cys(4)Fe(2)S(2) ferredoxins are a class of small acidic proteins that typically act as electron shuttles between NAD(P)H-dependent reductases and monoxygenases, particularly cytochromes P450. Nuclear magnetic resonance assignments and detailed analysis of nuclear Overhauser effects permit the direct comparison of the functional C-terminal domains of three vertebrate-type ferredoxins, the mammalian adrenodoxin (Adx) and the bacterial ferredoxins putidaredoxin (Pdx) and terpredoxin (Tdx). In particular, homologous hydrogen-bonding networks involving a conserved basic residue (His 49 in Pdx, His 56 in Adx, Arg 49 in Tdx) are detailed. This hydrogen bond network appears to play a role in the mechanical transmission of redox-dependent conformational and dynamic changes from the iron-sulfur binding loop to the C-terminal domain. Hydrogen/deuterium exchange measurements have been made in Adx as a function of oxidation state for comparison with previous studies of Pdx and Tdx. The results of these measurements highlight the importance of the conserved basic residue in the linkage between oxidation state and protein dynamics. Finally, a series of mutations have been made in the C-terminal domain of Pdx, including one, Y51F, that disrupts the proposed hydrogen-bonding network without perturbing steric and hydrophobic interactions in the functional domain. Although the mutant is considerably destabilized with respect to wild-type Pdx, relatively unperturbed chemical shifts for residues near the site of the mutation and NOEs between water and Phe 51 suggest that the network is reconstituted with a solvent water in place of the tyrosine hydroxyl group in this mutant.