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Crystallographic studies of different membrane cytochrome P450 enzymes have provided examples of distinct structural conformations, suggesting protein flexibility. It has been speculated that conformational selection is an integral component of substrate recognition and access, but direct evidence of such substate interconversion has thus far remained elusive. In the current study, solution NMR revealed multiple and exchanging backbone conformations for certain structural features of the human steroidogenic cytochrome P450 17A1 (CYP17A1). This bifunctional enzyme is responsible for pregnenolone C17 hydroxylation, followed by a 17,20-lyase reaction to produce dehydroepiandrosterone, the key intermediate in human synthesis of androgen and estrogen sex steroids. The distribution of CYP17A1 conformational states was influenced by temperature, binding of these two substrates, and binding of the soluble domain of cytochrome b5 (b5). Notably, titration of b5 to CYP17A1·pregnenolone induced a set of conformational states closely resembling those of CYP17A1·17α-hydroxypregnenolone without b5, providing structural evidence consistent with the reported ability of b5 to selectively enhance 17,20-lyase activity. Solution NMR thus revealed a set of conformations likely to modulate human steroidogenesis by CYP17A1, demonstrating that this approach has the potential to make similar contributions to understanding the functions of other membrane P450 enzymes involved in drug metabolism and disease states.

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.

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 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 improve our understanding of 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-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 CX(5)R 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.

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 nuclear magnetic resonance (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.

Crystallographic studies of different membrane cytochrome P450 enzymes have provided examples of distinct structural conformations, suggesting protein flexibility. It has been speculated that conformational selection is an integral component of substrate recognition and access, but direct evidence of such substate interconversion has thus far remained elusive. In the current study, solution NMR revealed multiple and exchanging backbone conformations for certain structural features of the human steroidogenic cytochrome P450 17A1 (CYP17A1). This bifunctional enzyme is responsible for pregnenolone C17 hydroxylation, followed by a 17,20-lyase reaction to produce dehydroepiandrosterone, the key intermediate in human synthesis of androgen and estrogen sex steroids. The distribution of CYP17A1 conformational states was influenced by temperature, binding of these two substrates, and binding of the soluble domain of cytochrome b5 (b5). Notably, titration of b5 to CYP17A1·pregnenolone induced a set of conformational states closely resembling those of CYP17A1·17α-hydroxypregnenolone without b5, providing structural evidence consistent with the reported ability of b5 to selectively enhance 17,20-lyase activity. Solution NMR thus revealed a set of conformations likely to modulate human steroidogenesis by CYP17A1, demonstrating that this approach has the potential to make similar contributions to understanding the functions of other membrane P450 enzymes involved in drug metabolism and disease states.

The purpose of this work was to study the reaction kinetics between two model sulfenamide prodrugs of linezolid, N-(phenylthio)linezolid and N-[(2-ethoxycarbonyl)ethylthio]linezolid, with free thiol-containing proteins; commercial human serum albumin (HSA); a constitutively active mutant of the protein tyrosine phosphatase PRL-1 (PRL-1-C170S-C171S), a model protein; and diluted fresh human plasma. The reaction was followed by high-performance liquid chromatography, both for the loss of prodrug and appearance of linezolid, and at different pH values with molar excess of the proteins relative to the prodrugs. Pseudo first-order kinetics was observed. Consistent with earlier findings for the reaction between similar sulfenamides and small-molecule thiols, the reaction kinetics appeared to be consistent with thiolate attack at the sulfenamide bond to release the parent drug. The proteins reacted significantly slower on a molar basis than their small-molecule counterparts. It appears that proteins such as HSA may play a role in the in vivo conversion of sulfenamide prodrugs to their parent drug.