The crystal structure of calmodulin (CaM) bound to trifluoperazine (TFP) has been determined and refined to a resolution of 2.45 A. Only one TFP is bound to CaM, but that is sufficient to cause distortion of the central alpha-helix and juxtaposition of the N- and C-terminal domains similar to that seen in CaM-polypeptide complexes. The drug makes extensive contacts with residues in the C-terminal domain of CaM but only a few contacts with one residue in the N-terminal domain. The structure suggests that substrate binding to the C-terminal domain is sufficient to cause the conformational changes in calmodulin that lead to activation of its targets.

The structure of huIFN-alpha 2b provides an accurate model for analysis of the > 15 related type 1 interferon molecules. HuIFN-alpha 2b displays considerable structural similarity with muIFN-beta, interleukin-10 and interferon-gamma, which also bind related class 2 cytokine receptors. From these structural comparisons and numerous studies on the effects of mutations on biological activity, we have identified protein surfaces that appear to be important in receptor activation. This study also reveals the potential biological importance of the huIFN-alpha 2b dimer.

Ectromelia virus (ECTV) encodes an IFN-␥-binding protein (IFN-␥BPectromelia virus ͉ immunomodulator ͉ interferon ͉ cytokine ͉ complex E ctromelia virus (ECTV) is an orthopoxvirus that causes mousepox, which closely resembles the genetic and disease characteristics of the human pathogen variola virus (VARV), the causative agent of smallpox (1). The genomes of all orthopoxviruses, including ECTV and VARV, encode proteins required for viral replication, as well as soluble cytokine-and chemokine-binding proteins, which disrupt the activation and recruitment of immune cells responsible for host antiviral responses (2, 3). The severity of smallpox and mousepox has been attributed to the effectiveness of these immunomodulatory proteins, which in many instances, exhibit significant homology to cellular receptors, suggesting they were captured and adapted to subvert host immune responses during poxvirus evolution.All orthopoxviruses express IFN-␥-binding proteins (IFN␥BPs) that efficiently block IFN-␥-mediated signaling cascades responsible for activating potent antiviral defense mechanisms. The importance of IFN-␥ in viral pathogenesis is demonstrated by studies in C57BL/6 mice, in which depletion of IFN-␥ by monoclonal antibody treatment, or disruption of the signaling pathway through genetic means, transforms benign ECTV infection into a lethal one (4, 5). In addition, the ectromelia virus IFN-␥BP (IFN-␥BP ECTV ) has been shown to be a critical virulence factor in BALB/c mice, in which ECTV infections are lethal but infections with an ECTV mutant lacking a functional IFN-␥BP ECTV are not (6).Orthopoxvirus IFN-␥BPs are Ϸ270-aa proteins that share Ͼ90% sequence identity with one another and Ϸ20% sequence identity with the extracellular region of the cellular IFN-␥R1 chains, often called the cytokine receptor homology region (CRHR). In contrast to cellular IFN-␥R1s, which exhibit species-specific binding to their cognate ligand, IFN-␥BPs exhibit relaxed IFN-␥-binding specificity (7, 8) (e.g., IFN-␥BP ECTV binds human, murine, rabbit, and bovine IFN-␥). This functional difference may have facilitated opportunistic viral infections in multiple hosts during the evolution of the virus.In contrast to the CRHR, the C-terminal Ϸ60 aa of the IFN-␥BPs share no identifiable sequence similarity with cellular proteins. Recent studies suggest that the C terminus mediates oligomerization of the IFN-␥-binding domains (9). However, there are conflicting reports about the quaternary structure of the molecules. For example, IFN-␥BPs encoded by VACV-WR (Western Reserve) and myxoma virus (M-T7) have been reported to form dimers and trimers, respectively (10, 11). In contrast to these reports, we have demonstrated recently that IFN-␥BPs from ECTV and VACV-B8R (Copenhagen strain) adopt larger oligomers in solution, likely tetramers, which are critical for antagonizing IFN-␥ activity (9).To address the basic mechanisms of IFN-␥ antagonism by orthopoxvirus IFN-␥BPs, we determined the crystal structure of IFN-␥BP ECTV bound to human IFN-␥. IFN-␥...

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IL-10 is a dimeric cytokine that must engage its high-affinity cell surface receptor, IL-10R1, to induce multiple cellular activities. Here we report the 1.9 A crystal structure of an engineered IL-10 monomer (IL-10M1) in complex with a neutralizing Fab fragment (9D7Fab). 9D7Fab and IL-10R1 bind distinct nonoverlapping surfaces on IL-10M1. Antagonism of the IL-10M1/IL-10R1 interaction is the result of 9D7Fab-induced conformational changes in the CD loop of IL-10M1 that indirectly alter the structure of the IL-10R1 binding site. A single mutation (Ile87Ala) in the same CD loop region of the Epstein-Barr virus IL-10 (ebvIL-10) also reduces IL-10R1 binding affinity, suggesting that ebvIL-10 and 9D7Fab use similar allosteric mechanisms to modulate IL-10R1 affinity and biological activity.