Robust generation of IgG bispecific antibodies has been a long-standing challenge. Existing methods require extensive engineering of each individual antibody, discovery of common light chains, or complex and laborious biochemical processing. Here we combine computational and rational design approaches with experimental structural validation to generate antibody heavy and light chains with orthogonal Fab interfaces. Parental monoclonal antibodies incorporating these interfaces, when simultaneously co-expressed, assemble into bispecific IgG with improved heavy chain-light chain pairing. Bispecific IgGs generated with this approach exhibit pharmacokinetic and other desirable properties of native IgG, but bind target antigens monovalently. As such, these bispecific reagents may be useful in many biotechnological applications.

amide exchange ͉ ankyrin repeat ͉ transcription regulation ͉ protein dynamics ͉ protein folding

IkappaBalpha is the major regulator of transcription factor NF-kappaB function. The ankyrin repeat region of IkappaBalpha mediates specific interactions with NF-kappaB dimers, but ankyrin repeats 1, 5 and 6 display a highly dynamic character when not in complex with NF-kappaB. Using chemical denaturation, we show here that IkappaBalpha displays two folding transitions: a non-cooperative conversion under weak perturbation, and a major cooperative folding phase upon stronger insult. Taking advantage of a native Trp residue in ankyrin repeat (AR) 6 and engineered Trp residues in AR2, AR4 and AR5, we show that the cooperative transition involves AR2 and AR3, while the non-cooperative transition involves AR5 and AR6. The major structural transition can be affected by single amino acid substitutions converging to the "consensus" ankyrin repeat sequence, increasing the native state stability significantly. We further characterized the structural and dynamic properties of the native state ensemble of IkappaBalpha and the stabilized mutants by H/(2)H exchange mass spectrometry and NMR. The solution experiments were complemented with molecular dynamics simulations to elucidate the microscopic origins of the stabilizing effect of the consensus substitutions, which can be traced to the fast conformational dynamics of the folded ensemble.

Transcription complex components frequently show coupled folding and binding but the functional significance of this mode of molecular recognition is unclear. IkappaBalpha binds to and inhibits the transcriptional activity of NF-kappaB via its ankyrin repeat (AR) domain. The beta-hairpins in ARs 5-6 in IkappaBalpha are weakly-folded in the free protein, and their folding is coupled to NF-kappaB binding. Here, we show that introduction of two stabilizing mutations in IkappaBalpha AR 6 causes ARs 5-6 to fold cooperatively to a conformation similar to that in NF-kappaB-bound IkappaBalpha. Free IkappaBalpha is degraded by a proteasome-dependent but ubiquitin-independent mechanism, and this process is slower for the pre-folded mutants both in vitro and in cells. Interestingly, the pre-folded mutants bind NF-kappaB more weakly, as shown by both surface plasmon resonance and isothermal titration calorimetry in vitro and immunoprecipitation experiments from cells. One consequence of the weaker binding is that resting cells containing these mutants show incomplete inhibition of NF-kappaB activation; they have significant amounts of nuclear NF-kappaB. Additionally, the weaker binding combined with the slower rate of degradation of the free protein results in reduced levels of nuclear NF-kappaB upon stimulation. These data demonstrate clearly that the coupled folding and binding of IkappaBalpha is critical for its precise control of NF-kappaB transcriptional activity.

Like most extracellular bacterial proteases, Streptomyces griseus protease B (SGPB) and ␣-lytic protease (␣LP) are synthesized with covalently attached pro regions necessary for their folding. In this article, we characterize the folding free energy landscape of SGPB and compare it to the folding landscapes of ␣LP and trypsin, a mammalian homolog that folds independently of its zymogen peptide. In contrast to the thermodynamically stable native state of trypsin, SGPB and ␣LP fold to native states that are thermodynamically marginally stable or unstable, respectively. Instead, their apparent stability arises kinetically, from unfolding free energy barriers that are both large and highly cooperative. The unique unfolding transitions of SGPB and ␣LP extend their functional lifetimes under highly degradatory conditions beyond that seen for trypsin; however, the penalty for evolving kinetic stability is remarkably large in that each factor of 2.4-8 in protease resistance is accompanied by a cost of ∼10 5 in the spontaneous folding rate and ∼5-9 kcal/mole in thermodynamic stability. These penalties have been overcome by the coevolution of increasingly effective pro regions to facilitate folding. Despite these costs, kinetic stability appears to be a potent mechanism for developing native-state properties that maximize protease longevity.Keywords: Streptomyces griseus protease B; protein folding; pro region; kinetic stability; protein evolution Supplemental material: See www.proteinscience.org Extracellular bacterial proteases, as well as vacuolar and lysosomal proteases, are generally synthesized with covalently attached pro regions required for their folding. Studies on ␣-lytic protease (␣LP) revealed that its pro region acts as a folding catalyst, accelerating the conversion of an on-pathway molten-globule folding intermediate to the native state from a t 1/2 of 1800 years to seconds ; Fig. 1). This catalysis of ␣LP folding requires two discrete functions of its pro region: stabilization of the folding transition state, which accelerates folding by a factor of 3 × 10 9 , and tight binding to the native state (K i ‫ס‬ 0.32 nM) (Peters et al. 1998), which drives the folding reaction toward the folded protease. Once folding is complete, the pro region is proteolytically degraded, releasing mature, native protease (Cunningham and Agard 2003). Interestingly, the ␣LP native state (Nat ␣LP ) is thermodynamically less stable than either the folding intermediate or the fully unfolded molecule. Only a large and highly cooperative unfolding barrier (t 1/2 ‫ס‬ 1.2 years) prevents the active protease from spontaneously unfolding. Recent experiments suggest that such kinetic stability proReprint requests to: David A. Agard, Howard Hughes Medical Institute and the Department of Biochemistry and Biophysics, University of California, San Francisco, 600 16th Street, Room S412, San Francisco, CA 94143-2240, USA; e-mail: agard@msg.ucsf.edu; fax: (415) .Abbreviations: SGPB, Streptomyces griseus protease B; ␣LP, ␣-lytic protease...