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Insulin-degrading enzyme (IDE) hydrolyzes monomeric polypeptides, including amyloid-β (Aβ) and HIV-1 p6. It also acts as a nonproteolytic chaperone to prevent Aβ polymerization. Here we compare interactions of Aβ and non-amyloidogenic p6 with IDE. Although both exhibited similar proteolysis rates, the binding kinetics to an inactive IDE characterized using relaxation-based NMR were remarkably different. IDE and Aβ formed a sparsely populated complex with a lifetime of milliseconds in which a short hydrophobic cleavage segment of Aβ was anchored to IDE. Strikingly, a second and more stable complex was significantly populated with a subsecond lifetime owing to multiple intermolecular contacts between Aβ and IDE. By selectively sequestering Aβ in this nonproductive complex, IDE likely increases the critical concentration required for fibrillization. In contrast, IDE and p6 formed a transient, submillisecond complex involving a single anchoring p6 motif. Modulation of intermolecular interactions, thus, allows IDE to differentiate between non-amyloidogenic and amyloidogenic substrates.

There is a significant need for new therapeutics to treat infections caused by the biodefense agent Bacillus anthracis. In pursuit of drug discovery against this organism, we have developed novel propargyl-linked inhibitors that target the essential enzyme dihydrofolate reductase (DHFR) from B. anthracis. Previously, we reported an initial series of these inhibitors and a high-resolution crystal structure of the ternary complex of the enzyme bound to its cofactor and one of the most potent inhibitors, UCP120B [Beierlein, J., Frey, K., Bolstad, D., Pelphrey, P., Joska, T., Smith, A., Priestley, N., Wright, D., and Anderson, A. (2008) J. Med. Chem. 51, 7532-7540]. Herein, we describe a three-dimensional solution structure of the ternary complex as determined by NMR. A comparison of this solution structure to the crystal structure reveals a general conservation of the DHFR fold and cofactor interactions as well as differences in the location of an active site helix and specific ligand interactions. In addition to data for the fully assigned ternary complex, data for the binary (enzyme-cofactor) complex were collected, providing chemical shift comparisons and revealing perturbations in residues that accommodate ligand binding. Dynamics of the protein, measured using (15)N T(1) and T(2) relaxation times and {(1)H}-(15)N heteronuclear NOEs, reveal residue flexibility at the active site that explains enzyme inhibition and structure-activity relationships for two different series of these propargyl-linked inhibitors. The information obtained from the solution structure regarding active site flexibility will be especially valuable in the design of inhibitors with increased potency.

A transmembrane glycoprotein member of the immunoglobulin superfamily of cell adhesion molecules (IgCAM),1 L1 is essential for many developmental processes. Mutation of the single gene encoding L1 in humans results in a number of devastating neurological abnormalities and mental retardation syndromes (1). L1 involvement in the metastatic progression has been well documented, and it is now considered an important target for treating specific tumor types, including ovarian carcinomas (2). The importance of understanding L1-CT oligomeric state comes from the notion that L1-mediated cell adhesion is associated with the activation of the MAP-kinase signaling pathway (3), the initial stages of which are characterized by L1 clustering. There are many classic examples, such as avidity modulation in integrins (4, 5), showing that receptor oligomerization is an important initial step during activation. Any part of the receptor may be involved in this process. Recent studies indicate that the third fibronectin type III domain of the L1 extracellular domain spontaneously homomultimerizes, leading to the formation of trimeric L1 and the concomitant recruitment of integrins (6). The transmembrane domain of human L1 contains a potential homooligomerization motif, GXXXG (7). However its role in L1 clustering has yet to be investigated. In this work we present evidence that the cytoplasmic domain of L1 is mostly monomeric in aqueous solution. In the nervous system L1 mediates cell migration, axon extension, branching, fasciculation, guidance, and interactions with glia (8, 9). Neuronal L1-CT (sequence presented in Figure 1A) contains four additional amino acids (1177RSLE) compared to L1-CT expressed in nonneuronal cells or in a variety of human tumors (10, 11). These are coded for by the alternatively spliced exon 27. Although several L1-associated cytosolic molecules participating in the axon growth and branching have been identified (12–14), the structural basis for L1-mediated intracellular signaling and cell remodeling still remains a mystery. Here we present biophysical and biochemical data characterizing free L1-CT in aqueous solution and in complexes with two of its binding partners, the ezrin FERM domain and the AP2-μ2 chain. Figure 1 (A) Sequences of L1-CT and the peptides derived from it. Three major regions, which are involved in ezrin binding, are underscored. (B) Differences between 13Cα/13Cβ shifts of each residue from the free L1-CT in aqueous solution and the ... The involvement of L1 in regulation of the axonal outgrowth and the neuronal migration requires coordination with the actin cytoskeleton. IgCAMs, such as L1, do not bind directly to the actin cytoskeleton; their interactions are regulated by MCLs. Ezrin, radixin, and moesin (ERM) form a family of highly homologous proteins that are ideal candidates for coordinating these interactions between L1 and the actin cytoskeleton during the axonal outgrowth and migration. ERM proteins have a C-terminal actin binding site (15) and an N-terminal FERM (f our point one, ezrin, radixin, and moesin, common to all members of the band 4.1 superfamily (16)) domain that can bind to the transmembrane molecules. Direct interactions between L1 and ezrin have been identified in vitro and confirmed in primary hippocampal neurons and nerve growth factor-treated PC12 cells (14). The L1-CT interaction with the ezrin FERM domain plays a crucial role in neurite branching (9) and involves two major sites: the juxtaproximal 1147KGGKYS-VKDK homologue of the nonpolar RxxTYxVxxA motif of ICAM2 (17) and the 1176YRSLE region (14). In this study we have confirmed these findings by NMR. Moreover, we have identified an additional region, which has previously been associated with L1 binding to another MCL, ankyrin (18). L1-controlled endocytosis is implicated in the motility of the nerve growth cones (19). The neuronal 1176YRSLE motif coordinates L1 endocytosis via clathrin-coated pits (13) via a tyrosine-based sorting signal (YxxΦ, where Φ is a residue with a bulky hydrophobic side chain) found in many other proteins (20): the 1176YRSL sequence of L1 serves as a docking site for the μ2 chain of the clathrin-associated AP2 complex (21). Along with this motif, an additional hydrophobic residue located three amino acids upstream (1176Y-3; 1173F in L1) is anticipated to be involved in this complex formation, as has been shown for the cytoplasmic domain of human P-selectin (22). In this work we have confirmed by NMR that the 1173FxxYxxL motif of L1 is indeed a major AP2-μ2 binding site. Our data, however, suggest that the L1-CT and AP2-μ2 interaction requires a longer motif, 1170D–1182D, which encompasses the above-mentioned residues.

N-Terminal self-cleavage (autoprocessing) of the HIV-1 protease precursor is crucial for liberating the active dimer. Under drug pressure, evolving mutations are predicted to modulate autoprocessing, and the reduced catalytic activity of the mature protease (PR) is likely compensated by enhanced conformational/dimer stability and reduced susceptibility to self-degradation (autoproteolysis). One such highly evolved, multidrug resistant protease, PR20, bears 19 mutations contiguous to sites of autoproteolysis in retroviral proteases, namely clusters 1-3 comprising residues 30-37, 60-67, and 88-95, respectively, accounting for 11 of the 19 mutations. By systematically replacing corresponding clusters in PR with those of PR20, and vice versa, we assess their influence on the properties mentioned above and observe no strict correlation. A 10-35-fold decrease in the cleavage efficiency of peptide substrates by PR20, relative to PR, is reflected by an only ∼4-fold decrease in the rate of Gag processing with no change in cleavage order. Importantly, optimal N-terminal autoprocessing requires all 19 PR20 mutations as evaluated in vitro using the model precursor TFR-PR20 in which PR is flanked by the transframe region. Substituting PR20 cluster 3 into TFR-PR (TFR-PR(PR20-3)) requires the presence of PR20 cluster 1 and/or 2 for autoprocessing. In accordance, substituting PR clusters 1 and 2 into TFR-PR20 affects the rate of autoprocessing more drastically (>300-fold) compared to that of TFR-PR(PR20-3) because of the cumulative effect of eight noncluster mutations present in TFR-PR20(PR-12). Overall, these studies imply that drug resistance involves a complex synchronized selection of mutations modulating all of the properties mentioned above governing PR regulation and function.

Over the last two decades, our knowledge concerning intracellular events that regulate integrin’s affinity to their soluble ligands has significantly improved. However, the mechanism of adhesion-induced integrin clustering and development of focal complexes, which could further mature to form focal adhesions, still remains under-investigated. Here we present a structural model of tandem IgC2 domains of skelemin in complex with the cytoplasmic tails of integrin αIIbβ3. The model of tertiary assembly is generated based upon NMR data and illuminates a potential link between the essential cell adhesion receptors and myosin filaments. This connection may serve as a basis for generating the mechanical forces necessary for cell migration and remodeling.