Background: Crystallography provides a static structure of cytochrome P450 17A1 (CYP17A1). Results: Solution NMR reveals an ensemble of CYP17A1 conformational substates. Conclusion: Ligand, cytochrome b 5 , or temperature alters the conformational CYP17A1 substates present. Significance: Changes in conformations probably modulate human steroidogenesis by CYP17A1.

The ability of a small molecule to bind and modify the activity of a protein target at a specific site greatly impacts the success of drugs in the pharmaceutical industry. One of the most important tools for evaluating these interactions has been high-field solution NMR because of its unique ability to examine even weak protein-drug interactions at high resolution. NMR can be used to evaluate the structural, thermodynamic and kinetic aspects of a binding reaction. The basis of NMR screening experiments is that binding causes a perturbation in the physical properties of both molecules. Unique properties of small and macromolecules allow selective detection of either the protein target or ligand, even in a mixture of compounds. This review outlines current methodologies for assessing protein-ligand interactions from the perspectives of the protein target and ligand and delineates the fundamental principles for understanding NMR approaches in drug research. Advances in instrumentation, pulse sequences, isotopic labeling strategies, and the development of competition experiments support the study of higher molecular weight protein targets, facilitate higher-throughput and expand the range of binding affinities that can be evaluated, enhancing the utility of NMR for identifying and characterizing potential therapeutics to druggable protein targets.

Phosphatase of regenerating liver-1 (PRL-1) belongs to a unique subfamily of protein tyrosine phosphatases (PTPases) associated with oncogenic and metastatic phenotypes. While considerable evidence exists to supports a role for PRL-1 in promoting proliferation, the biological regulators and effectors of PRL-1 activity remain unknown. PRL-1 activity is inhibited by disulfide bond formation at the active site in vitro, suggesting PRL-1 may be susceptible to redox regulation in vivo. Because PRL-1 has been observed to localize to several different subcellular locations and cellular redox conditions vary with tissue type, age, stage of cell cycle and subcellular location, we determined the reduction potential of the active site disulfide bond that controls phosphatase activity to better understand the function of PRL-1 in various cellular environments. We used high-resolution solution NMR spectroscopy to measure the potential and found it to be −364.3 ± 1.5 mV. Because normal cellular environments range from −170 to −320 mV, we concluded that nascent PRL-1 would be primarily oxidized inside cells. Our studies show that a significant conformational change accompanies activation, suggesting a post-translational modification may alter the reduction potential, conferring activity. We further demonstrate that alteration of the C-terminus renders the protein reduced and active in vitro, implying the C-terminus is an important regulator of PRL-1 function. These data provide a basis for understanding how subcellular localization regulates the activity of PRL-1 and, with further investigation, may help reveal how PRL-1 promotes unique outcomes in different cellular systems, including proliferation in both normal and diseased states.Phosphatase of Regenerating Liver (PRL) enzymes are a unique subfamily of protein tyrosine phosphatases (PTPases) and play an important role in maintaining appropriate tyrosine phosphorylation levels in the cell during development and tissue regeneration (1). This subfamily includes three homologous members (PRL-1, −2 and −3) that share a high degree of amino acid sequence identity (>75%). PRL-1 (20 kDa) was first discovered in regenerating liver following partial hepatectomy as the product of an early-immediate response gene (2) and has since been implicated in the repair of other tissues including neurons, oligodendrocytes and the cerebral cortex in response to transient forebrain ischemia (3). PRLs are normally expressed at low levels in most mature cells, while higher expression levels are observed during embryonic development (4) and during cellular proliferation (5-9) or differentiation (5,10, † This publication was made possible by NIH grant number P20 RR-17708 from the National Center for Research Resources and the Kansas University Center for Research. This work was additionally supported by fellowships for Andria Skinner from Amgen and the Edith and Eleta Ernst Cancer Research Fellowship. The Q-Tof2tm was purchased with support from KSTAR, Kansas administered NSF EPSCoR and the U...

Phosphatase of regenerating liver-1 (PRL-1) is a novel target for potentially treating cancer metastases. Although its specific biochemical role in these processes has yet to be delineated, considerable evidence suggests the phosphatase activity of PRL-1 is required for promoting cancer and metastasis. PRL-1 belongs to the protein tyrosine phosphatase (PTPase) family and functions using the CX5R consensus active site motif. Like other PTPases, PRL-1 is inhibited by oxidation at its active site Cys, however, disulfide bond formation occurs unusually readily in wild-type PRL-1. Chemical shift assignments are available for oxidized wild type, but numerous, substantial changes are observed in the spectra upon reduction. Because the reduced form is active, we sought to identify a stable mutant that would resist oxidation and be useful for facilitating drug screening and development using NMR-based assays. We present here NMR assignments for a full-length, reduced and active form of PRL-1, PRL-1-C170S-C171S, that is well suited for this purpose.

It is well established that the oxidation state of cysteine residues in proteins are critical to overall physical stability. The presence of disulfide bonds most often imparts thermodynamic stability, and as such, engineered disulfide bonds have become a means for improving the viability of protein therapeutics. In some cases, however, disulfide bonds can diminish stability. Because proteins are held together by numerous weak interactions, understanding the mechanisms by which stabilization is achieved is important to the design of new biotechnology products that better resist unfolding and aggregation. Mechanistic information describing how specific interactions influence stability is lacking, in part because the techniques typically used to study inherent stability do not provide sufficient detail. In the present study, a model protein system, phosphatase of regenerating liver (PRL-1), was used to investigate the role of cysteine residues on physical stability. A combination of chemical modulation and mutagenesis was employed to alter the redox state of the protein, and the effects were observed using a combination of low- and high-resolution methods. Specifically, solution NMR data revealed the stability of PRL-1 depends on cooperation between local interactions with the Cys side chains. This approach provides a means to better understand how protein stabilization is achieved.