The CD147 receptor plays an integral role in numerous diseases by stimulating the expression of several protein families and serving as the receptor for extracellular cyclophilins; however, neither CD147 nor its interactions with its cyclophilin ligands have been well characterized in solution. CD147 is a unique protein in that it can function both at the cell membrane and after being released from cells where it continues to retain activity. Thus, the CD147 receptor functions through at least two mechanisms that include both cyclophilin-independent and cyclophilin-dependent modes of action. In regard to CD147 cyclophilin-independent activity, CD147 homophilic interactions are thought to underlie its activity. In regard to CD147 cyclophilin-dependent activity, cyclophilin/CD147 interactions may represent a novel means of signaling since cyclophilins are also peptidyl–prolyl isomerases. However, direct evidence of catalysis has not been shown within the cyclophilin/CD147 complex. In this report, we have characterized the solution behavior of the two most prevalent CD147 extracellular isoforms through biochemical methods that include gel-filtration and native gel analysis as well as directly through multiple NMR methods. All methods indicate that the extracellular immunoglobulin-like domains are monomeric in solution and, thus, suggest that CD147 homophilic interactions in vivo are mediated through other partners. Additionally, using multiple NMR techniques, we have identified and characterized the cyclophilin target site on CD147 and have shown for the first time that CD147 is also a substrate of its primary cyclophilin enzyme ligand, cyclophilin A.

SUMMARY Many protein systems rely on coupled dynamic networks to allosterically regulate function. However, the broad conformational space sampled by non-coherently dynamic systems has precluded detailed analysis of their communication mechanisms. Here, we have developed a methodology that combines the high sensitivity afforded by nuclear magnetic resonance relaxation techniques and single-site multiple mutations, termed RASSMM, to identify two allosterically coupled dynamic networks within the non-coherently dynamic enzyme cyclophilin A. Using this methodology, we discovered two key hotspot residues, Val6 and Val29, that communicate through these networks, the mutation of which altered active-site dynamics, modulating enzymatic turnover of multiple substrates. Finally, we utilized molecular dynamics simulations to identify the mechanism by which one of these hotspots is coupled to the larger dynamic networks. These studies confirm a link between enzyme dynamics and the catalytic cycle of cyclophilin A and demonstrate how dynamic allostery may be engineered to tune enzyme function.

Biocatalysis in ionic liquids (ILs) is largely limited by the instabilities of enzymes in these solvents, thus negating their auspicious solvent properties. Here, we have engineered an IL-tolerant variant of lipase A (lipA) from Bacillus subtilis by examining the site-specific interactions of lipA with 1-butyl-3-methylimidazolium chloride ([BMIM]- [Cl]). Results of NMR analysis found that [BMIM][Cl] induced structural perturbations near the active site of lipA, underscoring the importance of mediating direct ion interactions with the IL in this region. Mutation of G158 near the active site to glutamic acid resulted in a 2.5-fold improvement in tolerance of lipA to 2.9 M (or 50% v/v) [BMIM][Cl], which correlated with the retention of active site structure. The effect of the G158E mutation was likely the result of inhibition of hydrophobic interactions with the [BMIM] cation. Further analysis of the electrostatic surface of lipA led to the mutation of K44 to glutamic acid to diminish the attraction of chloride anions, which also improved lipA tolerance to [BMIM][Cl]. Beneficial point mutations, which had an additive effect on lipA stability, were combined, resulting in a super stable lipA quadruple mutant (G158E/K44E/R57E/Y49E) with a 7-fold improvement in stability. In comparison, nonspecific charge modification via acetylation and succinylation resulted in only a 1.2and 1.9-fold improvement in stability, respectively, over wild-type lipA. Ultimately, these results, while providing insight into the nature of the structural effects of ILs on enzymes, highlight the utility of combining NMR and charge engineering to rationally optimize enzyme stability for biocatalysis in ILs.

With the recent advances in NMR relaxation techniques, protein motions on functionally important timescales can be studied at atomic resolution. Here, we have used NMR-based relaxation experiments at several temperatures and both 600 and 900 MHz to characterize the inherent dynamics of the enzyme cyclophilin-A (CypA). We have discovered multiple chemical exchange processes within the enzyme that form a ''dynamic continuum'' that spans 20-30 Å comprising active site residues and residues proximal to the active site. By combining mutagenesis with these NMR relaxation techniques, a simple method of counting the dynamically sampled conformations has been developed. Surprisingly, a combination of point mutations has allowed for the specific regulation of many of the exchange processes that occur within CypA, suggesting that the dynamics of an enzyme may be engineered.

Thermophilic proteins have found extensive use in research and industrial applications because of their high stability and functionality at elevated temperatures while simultaneously providing valuable insight into our understanding of protein folding, stability, dynamics, and function. Cyclophilins, constituting a ubiquitously expressed family of peptidyl–prolyl isomerases with a range of biological functions and disease associations, have been utilized both for conferring stress tolerances and in exploring the link between conformational dynamics and enzymatic function. To date, however, no active thermophilic cyclophilin has been fully biophysically characterized. Here, we determine the structure of a thermophilic cyclophilin (GeoCyp) from Geobacillus kaustophilus, characterize its dynamic motions over several time scales using an array of methodologies that include chemical shift-based methods and relaxation experiments over a range of temperatures, and measure catalytic activity over a range of temperatures to compare its structure, dynamics, and function to those of a mesophilic counterpart, human cyclophilin A (CypA). Unlike those of most thermophile/mesophile pairs, GeoCyp catalysis is not substantially impaired at low temperatures as compared to that of CypA, retaining ~70% of the activity of its mesophilic counterpart. Examination of substrate-bound ensembles reveals a mechanism by which the two cyclophilins may have adapted to their environments through altering dynamic loop motions and a critical residue that acts as a clamp to regulate substrate binding differentially in CypA and GeoCyp. Fast time scale (pico- to nanosecond) dynamics are largely conserved between the two proteins, in accordance with the high degree of structural similarity, although differences do exist in their temperature dependencies. Slower (microsecond) time scale motions are likewise localized to similar regions in the two proteins with some variability in their magnitudes yet do not exhibit significant temperature dependencies in either enzyme.

Tubulin is important for a wide variety of cellular processes including cell division, ciliogenesis, and intracellular trafficking. To perform these diverse functions, tubulin is regulated by post-translational modifications (PTM), primarily at the C-terminal tails of both the α- and β-tubulin heterodimer subunits. The tubulin C-terminal tails are disordered segments that are predicted to extend from the ordered tubulin body and may regulate both intrinsic properties of microtubules and the binding of microtubule associated proteins (MAP). It is not understood how either interactions with the ordered tubulin body or PTM affect tubulin’s C-terminal tails. To probe these questions, we developed a method to isotopically label tubulin for C-terminal tail structural studies by NMR. The conformational changes of the tubulin tails as a result of both proximity to the ordered tubulin body and modification by mono- and polyglycine PTM were determined. The C-terminal tails of the tubulin dimer are fully disordered and, in contrast with prior simulation predictions, exhibit a propensity for β-sheet conformations. The C-terminal tails display significant chemical shift differences as compared to isolated peptides of the same sequence, indicating that the tubulin C-terminal tails interact with the ordered tubulin body. Although mono- and polyglycylation affect the chemical shift of adjacent residues, the conformation of the C-terminal tail appears insensitive to the length of polyglycine chains. Our studies provide important insights into how the essential disordered domains of tubulin function.

Telomerase is essential for continuous cellular proliferation. Substantial insights have come from studies of budding yeast telomerase, which consists of a catalytic core in association with two regulatory proteins, ever shorter telomeres 1 and 3 (Est1 and Est3). We report here a high-resolution structure of the Est3 telomerase subunit determined using a recently developed strategy that combines minimal NMR experimental data with Rosetta de novo structure prediction algorithms. Est3 adopts an overall protein fold which is structurally similar to that adopted by the shelterin component TPP1. However, the characteristics of the surface of the experimentally determined Est3 structure are substantially different from those predicted by prior homology-based models of Est3. Structure-guided mutagenesis of the complete surface of the Est3 protein reveals two adjacent patches on a noncanonical face of the protein that differentially mediate telomere function. Mapping these two patches on the Est3 structure defines a set of shared features between Est3 and HsTPP1, suggesting an analogous multifunctional surface on TPP1.RASREC Rosetta | OB-fold protein T elomerase is a telomere-dedicated DNA polymerase that is responsible for telomere-length maintenance in most eukaryotes. In cells that lack telomerase, gradual erosion due to incomplete replication of duplex telomeric DNA leads to an eventual block to cellular proliferation. Ectopic expression of telomerase in human cells is sufficient to confer cellular immortality (1) and it is up-regulated in over 90% of tumor biopsies (2). Conversely, reduced telomerase activity is responsible for the age-dependent effects on organs that rely on continual replenishment throughout a normal human life span and can lead to bone marrow failure, pulmonary fibrosis, or aplastic anemia (3). Hence, an increased understanding of the roles of telomerase and its accessory proteins in telomere length homeostasis has the potential to impact several different aspects of human health.The yeast telomerase holoenzyme is composed of three proteins [the catalytic ever shorter telomere 2 (Est2) subunit, along with the Est1 and Est3 regulatory proteins], which together form a complex with the TLC1 RNA (4, 5). In vivo, telomerase is highly regulated, in that only a subset of telomeres are elongated in each cell cycle (6); however, the mechanism that restricts telomerase to a limited number of substrates is still poorly understood. This deficit stems at least in part from the fact that the surface of yeast telomerase represents a largely unexplored territory. Even though there are numerous interaction surfaces on the three Est proteins with the potential to regulate important interactions, the only well-characterized regulatory step involving Est proteins is the recruitment of telomerase to the telomere through the direct interaction of Est1 and the end-binding protein Cdc13 (7), which was originally uncovered using a labor-intensive genetic approach (8).High-resolution structural information provides a str...

No abstract