Posttranslational Chemical Mutagenesis - American Chemical Society

Oct 3, 2018 - Centro de Biologia Molecular Estrutural, Departamento de Bioquímica, Universidade Federal de Santa Catarina, 88040-970. Florianópolis,...
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Posttranslational chemical mutagenesis: to reveal the role of noncatalytic cysteine residues in pathogenic bacterial phosphatases Jean Bertoldo, Hernán Terenzi, Stefan Hüttelmaier, and Gonçalo J.L. Bernardes Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00639 • Publication Date (Web): 03 Oct 2018 Downloaded from http://pubs.acs.org on October 3, 2018

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Biochemistry

Posttranslational chemical mutagenesis: to reveal the role of noncatalytic cysteine residues in pathogenic bacterial phosphatases Jean B. Bertoldo,*1,2 Hernán Terenzi,3 Stefan Hüttelmaier,2 and Gonçalo J. L. Bernardes*1,4 1Department

of Chemistry, University of Cambridge, Lensfield Road, CB2 1EW Cambridge, UK. für Molekulare Medizin, Medizinische Fakultät, Martin-Luther-Universität Halle-Wittenberg, KurtMothes-Stra 3a, Halle, Germany 3Centro de Biologia Molecular Estrutural, Departamento de Bioquímica, Universidade Federal de Santa Catarina, 88040-970, Florianópolis-SC, Brazil. 4Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Avenida Professor Egas Moniz, 1649-028, Lisboa, Portugal. 2Institut

ABSTRACT: The field of chemical site-selective modification of proteins has progressed extensively in recent decades to enable protein functionalization for imaging, drug-delivery and functional studies. In this perspective, we provide a detailed insight into an alternative use of site-selective protein chemistry to probe the role(s) of unpaired Cys residues in the structure and function of disease relevant proteins. Phosphatases are important players in the successful infection of pathogenic bacteria, which represent a significant health burden, particularly in multi-drug-resistant strains. Therefore, a strategy to readily probe key amino-acid role(s) in the structure and function may facilitate the targeting and inhibition of these virulence factors. With a dehydroalanine-based posttranslational chemical mutagenesis approach, it is possible to reveal hitherto unknown function(s) of non-catalytic Cys residues and confirm their role and interplay in pathogenic bacterial phosphatases. By selectively modifying reactive sulfhydryl side chains in different protein local environments, this posttranslational site-selective chemical mutagenesis approach reveals structural information about binding pockets and regulatory roles of the modified residues, which can be further validated by conventional sitedirected mutagenesis. Ultimately, these new binding pockets can serve as templates for enhanced structure-based drugdesign platforms and aid the development of potent and specific inhibitors.

Cysteine (Cys) provides key roles in protein function and stability even though it is one of the least abundant amino acids. Its appearance on the genetic code is rather late1 but its accumulation continues in modern organisms2. Cys residues have a unique set of chemical properties, such as the ability to form disulfide bridges and to operate as redox switches3 through the high nucleophilicity of the sulfhydryl side chain (R–SH) of Cys4. The thiol can be alkylated by electrophiles and oxidized by reactive oxygen (ROS) and nitrogen species (RNS)5, which presents a challenge to proteins that contain highly reactive Cys, hence their tightly regulated expression under oxidative stress6. As a safety mechanism many of these proteins express key Cys residues in buried motifs7 and to preserve protein activity,8 a feature that likely evolved under selective pressure. Pathogenic bacteria, however, employ enzymes with highly reactive and surfaced exposed Cys9, 10. These enzymes promote the successful establishment of bacterial infection by helping bacteria circumvent host defenses.11-13 In addition, the cell host defense mechanism also involves a burst of ROS, that can lead to overoxidation, nevertheless these enzymes manage to avoid irreversible inactivation13 by employing mechanisms that involve catalytic and noncatalytic Cys. For example, the catalytic sulfur atom on

Cys can react with a nearby nitrogen atom to form a sulfenylamide14. A non-catalytic Cys, usually referred to as “backdoor” residue, can form a reversible disulfide bond with the catalytic Cys. This bond can also be formed between two non-catalytic backdoor Cys residues as seen in several phosphatases15-18 including the low molecular weight protein tyrosine phosphatase family (LMWPTPs)19. Overall, these non-catalytic Cys residues can be found in pairs, clusters, in single or multiple places within the protein structure. They can function as redox switches and have enhanced interplay when clustered, which provides a suitable environment to prevent irreversible inactivation20, 21. These features point to an elaborate, synergistic interplay amongst Cys residues in bacterial phosphatases, especially with non-catalytic residues. It also advocates for an in-depth analysis of their role on virulence effectors of pathogenic bacteria. The reactivity and localization of these non-catalytic Cys residues can be further exploited for drug design approaches as their role may prove critical for the maintenance of Cys-containing proteins18. As a matter of fact, non-catalytic Cys residues have already proven to be effective targets in FDAapproved drugs like the kinase-targeting ibrutinib22. However, the crucial point lies in the identification of

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which Cys residues merit further investigation, therefore, a strategy to readily reveal their roles in protein structure and activity may better inform the design of new antibacterial drugs. In this context, we seek to demonstrate how a recently developed method, which uses a chemical mutagenesis approach, can be confidently used to shed light on the role of non-catalytic Cys residues in bacterial enzymes and facilitate the discovery of suitable targets for antibacterial therapeutics. Our main goal is not to exhaustively describe and discuss the reactions developed so far for chemical site-selective protein modification that can be found in recent reviews23-27, but rather, we will focus on the potential and progress of such reactions in “posttranslational chemical mutagenesis”,28-30 a meth-

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odology that enables the modification of specific residues within the protein target, which permits the incorporation of synthetic modifications to explore amino-acid side-chain diversity31, 32. This Perspective will give detailed insight into this exciting emerging area and provide robust information on an alternative use; to reveal the role(s) of unpaired Cys residues in the structure and function of disease relevant proteins to advocate, hopefully, for its addition to the current chemical biology toolkit used to probe Cys reactivity (Figure 1). First in bacterial phosphatases and ultimately to study the interplay between Cys residues in a diverse panel of multiple Cys-containing proteins relevant in disease.

Figure 1. Overview of a posttranslational mutagenesis approach to probe amino-acid sequence, structure and activity of multiple Cys-containing proteins. A chemical probe, in this case α,α’-di-bromo-adipyl(bis)amide, designed to react with free Cys residues and convert these into Dha is incubated with the respective native Cys-protein. PTM protein Dha-protein is assessed for enzymatic activity or binding activity. Once the effect on activity or structure is assigned, the preferentially modified residue(s) are identified by mass spectrometry. A single plasmid construct that contains an Ala or Ser substitution of the preferentially modified residue(s) is cloned and expressed. The recombinant mutant protein is assessed and the role of the modified residues in the structure (by circular dichroism for secondary structural content and, for example, by molecular dynamics simulations for local structural changes) and activity is confirmed. For the molecular dynamics simulations: structural ensembles derived from 0.5 µs MD simulations on chemical mutant Dha53 of PtpA. Both the global 3D shape and the local structure of the protein is retained upon chemical mutation. Cys 11 and 16 of the active site are also shown.

PROBING CYS REACTIVITY IN PTPs Proteome-wide analysis As a result of the susceptibility of the thiol side chain to ROS, and its involvement in the direct regulation of PTPs33, the first methods to probe Cys reactivity were

developed to detect oxidized PTPs34. The most common, indirect techniques rely on the ability of active PTPs to react stoichiometrically and irreversibly with alkylating agents, i.e. iodoacetic acid (IAA) or N-ethylmaleimide and the resistance of oxidized PTPs to alkylation. Generally, cells are lysed in the presence of a labelled alkylat-

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Biochemistry

ing agent (e.g. radioactive) and oxidation is monitored upon decrease of detection of that probe. Oxidized PTPs are converted into the active state (by using reducing agents), captured and detected by using a PTP-reactive probe (e.g. biotin-tagged IAA)35. Although effective at detecting oxidized PTPs, these approaches failed to depict other reactive non-catalytic Cys because the outcome depends mostly on enzyme activity (e.g. in-gel phosphatase assay, IAP-biotin and activity-based probes ABP36). In addition, the alkylating agent added to the reaction mixture invariably modifies all reduced Cys except the oxidized residue, which prevents an understanding of intrinsic Cys reactivity and their interplay. Furthermore, the antibodies used to detected oxidized PTPs in proteomic analysis are raised against the conserved PTP signature motif VHCSO3HSAG, which covers only the catalytic cleft37. Although useful, this approach only probes oxidized catalytic Cys. This caveat is also present in the analysis of recombinantly expressed PTPs, which makes the study of the interplay between Cys residues difficult. However, over the past few years, more direct and improved approaches have been developed (recently reviewed38), based on Cys-reactive probes, which enabled the analysis of highly reactive Cys regardless of their catalytic nature10. These new approaches were based on the pioneering work by Cravatt and co-workers39. The authors developed an activity-based protein profiling probe called isoTOP-ABPP (isotopic tandem orthogonal proteolysis-activity-based protein profiling) that, combined with LC-MS/MS analysis, allowed the identification of highly reactive non-catalytic Cys residues in different proteomes. Their findings also revealed that some of the identified non-catalytic Cys residues possessed a previously unknown function and were significantly more conserved in eukaryotic organisms. Their findings provided strong evidence that non-catalytic Cys residues play essential roles in the proteome and merit special attention. Reasonably, proteome-wide reactivity profiling aids in the identification of highly reactive noncatalytic Cys, both in proteomic analysis and purified protein systems. However, the functional assignment of the identified residues still requires detailed kinetic and mutagenic experiments. In this scenario, we believe that our methodology may be useful in targeted chemical mutagenesis to study specific protein targets. If one multiple Cys-containing protein is selected, then no structural or activity information is available. Usually, to probe new functionalities, a previous putative role is assigned to the amino acids to be studied, based generally on conserved structural motifs identified through bioinformatics. Thus, consecutive rounds of cloning, expression and purification are required for each targeted mutation, which may result in a time-consuming, trial-and-error investigation. To facilitate inquiry into the functionality of new amino acids, a direct, chemistry-driven mutagenesis approach can be used to obtain otherwise unknown information about function and interplay of specific(s) amino acid(s). For example, by targeting the most reactive Cys within a protein with multiple Cys residues, suitable chemical probes used in a posttranslational chemical mutagenesis can provide new insights into the reactivity and role(s) of the preferentially modified residue(s). Sitedirected mutagenesis is employed because the residue(s) is identified by mass spectrometry. Upon in vitro con-

firmation, the role of the chemically modified residues can be further evaluated in transiently transfected cells, or stably, by expressing cell lines that contain the Cysmutant form of the protein target. This way a more biologically relevant protein activity or regulation can be uncovered. Posttranslational chemical mutagenesis Chemical biologists have been designing chemical probes for Cys modification since 1965 when Wilchek and colleagues demonstrated the conversion of serine (Ser) into Cys in small peptides40. Wilchek’s work demonstrated how the protein amide backbone and side chains could be exploited for selective chemical mutagenesis. Soon after, new milestones on chemical mutations on proteins were achieved (Table 1). Some of the first examples of chemical mutagenesis used conditions that were considered to be harsh for proteins. For example, in the pioneering work by Clark and Lowe, the nucleophilic Cys of papain was chemically converted into Ser by means of alkylation with phenacyl bromide, and subsequent photolysis and reduction with NaBH441. Basic or acidic conditions, high temperatures, organic solvents and other non-ideal conditions have been used continuously until recently. In addition, these reactions suffered from poor selectivity, which results in a mixture of modified products at different sites within the protein sequence. To avoid these drawbacks, improved reagents and site-selective chemical reactions that proceed efficiently under mild conditions were developed23-27. These new reactions proved to be reliable alternatives to maintain, or even extend, protein activity without disrupting how it folds; for instance, fluorescent-tagged proteins for imaging in which a precise position for modification is required,26, 27 antibody-drug conjugates used for cancer diagnostics, and to delivery payloads of antitumor drugs24, 42. In addition, these reactions enabled the chemoselective labelling of ribosomal-incorporated noncanonical amino acids that bear unnatural side chains, such as alkynes, alkenes, tetrazines, ketones and azides on endogenously expressed proteins43,44. These unusual side chains permitted bioorthogonal protein labelling and functionalization, which facilitates the detailed understanding of biological processes and biomolecules within their native habitat, cells and live organisms45. Another use of site-selective modification is the specific installation of suitable mimics of posttranslational modifications (PTMs) in recombinantly expressed proteins at defined positions to study and modulate enzyme activity. In a seminal work, Davis46 and colleagues engineered a Cys on the surface of kinase p38α. The same group has previously described the use of α,α’-di-bromoadipyl(bis)amide to selectively convert Cys into dehydroalanine (Dha)47. Subsequently, and through thiolMichael addition with sodium thiophosphate, a phospho-Cys mimic is installed46. The similarity of phosphoCys with naturally occurring phospho-Tyr was sufficient to switch the kinase to its active form. Several synthetic mimics of PTMs were facilitated by the ability to generate Dha in a site-selective way on proteins. A regioisomer of Histidine (His), iso-His (Hisiso), installed through β,ࢽC,N aza-Michael mutagenesis was used to probe the contributing roles of size and hydrogen bonding in the activ-

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ity of Mycobacterium tuberculosis (Mtb) pantothenate synthetase (PanC)48. His mimic H44Hisiso, which lacks the ability to donate H-bonds, provided the mechanistic detail for the inhibition of PanC. The chemical mutant preferentially adopted a rotamer that is oriented towards the solvent, which made it unable to interact with the βphosphate from ATP. PanC ATP-dependent condensation activity is important for the formation of vitamin B5 (panthothenate), which Mtb rely on to grow and for the proper function of its virulence factors49. Due to its important activity PanC is also a target for antituberculosis therapy. Interestingly, the same mimic Hisiso, installed into the protease subtilisin from Bacillus lentus (S156Hisiso), revealed that H-bond donors are not necessary to maintain enzymatic activity48. Acetylated and methylated Lys mimics were also produced by siteselective chemical mutagenesis and successfully used to study writer/erase activity of histone deacetylases (HDAC) in chromatin modification50. To date the acetylLys mimic installed through Dha in this study is the most convenient way to investigate HDAC activity on fulllength histones. Dha is currently used as a chemical precursor of mono-, di- and trimethylated Lys, acetylated Lys, phosphorylated Ser and glycosylated Ser, and has proven to be a reliable chemical handle to mimic PTMs and modulate enzymatic activity46. Interestingly, what the aforementioned approaches do not address are the reasons why nature has evolved to enhance the reactivity of particular side-chains within a protein sequence and what are the structural and functional consequences. Why, in a complex mixture of residues spread along the protein sequence, do some residues have enhanced reactivity that allows them to be preferentially chemically modified? Do they have a structural or functional role? Do physicochemical properties dictate their reactivity? Does reactivity mean that these

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residues perform a special role? The case is not simple for Cys, but it may provide insights into why a preferentially modified Cys in a multiple Cys-containing protein might play an important role in redox sensing, binding to an antigen or catalytic activity9. It is recognized that structural context dictates reactivity, and highly polarizable sulfhydryl side-chains are often presented in buried motifs8, which affects their accessibility and protonation state. Secondary structures, such as α-helices, can exhibit a dipole moment with a positive charge at the Nterminal end, which lowers the pKa and makes the Cys residues located within these constraints more reactive51. Such features enhance Cys nucleophilicity by reducing its pka from an “unperturbed” value of 8.5,52 to values as low as 3.553. In addition, some proteins have specialized catalytic pocket designs that also strongly enhance Cys reactivity towards specific substrates, such as peroxiredoxins54. Often, if there are two free Cys units, the reactivity of one is dependent on the other. Understanding Cys reactivity and interplay in this context might prove to be a challenge. To address this challenge, we developed an approach that combined a Dha-based chemical mutagenesis with site-directed mutagenesis. We hypothesized that Cys-toDha converter compound α,α’-di-bromoadipyl(bis)amide could react with highly reactive Cys in multiple Cys-containing bacterial phosphatases in accordance with Cys reactivity, which may highlight a possible key role. We then tested the Cys high reactivity/key function correlation by producing mutants of the preferentially modified residues and analysing the structure and enzymatic activity of both chemical and site-directed mutants (Figure 1). Our findings indicated that a Dhabased posttranslational chemical mutagenesis can be used as means to interrogate Cys function. We present here two case studies on which to base this assertion.

Table 1. Selected examples of chemical mutagenesis through Cys modification on proteins Year

Milestone

Compound/Methods

Use

Ref

1935

Cys Alkylation

Iodoacetamide

Peptide Mapping

55

1956

Cys–to–Lys Mutation

Aminoethyl

Activity Profiling

56

1956

Cys Crosslinking

Maleimide

Protein Conjugation

57

1965

Ser–to–Cys Mutation

Thioacetate

Peptide Modification

40

1977/78

Cys–to–Ser/Gly Mutation

α-Bromoacetophenone

Protein Modification

41, 58

1998

Disulfide Formation

Active Thiol

Protein Conjugation

59

2000

Disulfide Formation

Methanethiosulfonate

Protein Modification

60

2008

Cys–to–Dha sion

Mesitylenesulfonylhydroxylamine

Protein Conjugation

61

Conver-

CASE STUDY 1 – Non-catalytic Cys16 and Cys53 from Mycobacterium tuberculosis protein tyrosine phosphatase A (PtpA)

Our analysis of PtpA provides the first evidence to support the use of posttranslational chemical mutagenesis in revealing the critical role of non-catalytic Cys residues in bacterial phosphatases9. PtpA possesses three Cys residues, Cys11, Cys16 and Cys53, and its phospha-

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Biochemistry

tase activity relies on the nucleophilicity and reactivity of catalytic Cys11. As a result of PtpA dephosphorylation activity, Mtb is able to avoid the macrophage proteolysis machinery. PtpA disrupts key components of the macrophage endocytic pathway, which leads to the inhibition of phagosome maturation and its late fusion with the lysosome, which ultimately allows Mtb survival inside the host macrophages62, 63. As a result of its key activity, PtpA has become an important druggable target in antituberculosis chemotherapy64, 65. To probe Cys reactivity and function in PtpA we applied our posttranslational chemical mutagenesis approach (Figure 1). In this new approach in vitro reactions of recombinant PtpA and increasing equivalents of α,α’-di-bromoadipyl(bis)amide were performed under mild conditions (50 mM NaH2PO4, pH 8.0). The reaction resulted in a single protein population with one modified Cys after 1 h incubation with 75 equiv. of reagent at 37°C. Surprisingly, the selective single modification was achieved readily and without further optimization. Upon MS/MS analysis of the single modified protein digests, Cys53 residue (Dha53) was identified as being preferentially modified. Structural (circular dichroism and molecular dynamics) and functional assays (phosphatase activity) revealed that the conformation and phosphatase activity of the Dha53 mutant was not altered relative to the nonmodified PtpA. Subsequently, to corroborate the results from chemical mutagenesis, a site-directed C53A mutant was produced and its activity profile analyzed relative to the Dha53 mutant. As expected the C53A mutant presented the same phosphatase activity as the wild type and Dha53 mutant. Previously, Cys53 had been identified as a target of S-nitrosylation by nitric oxide66 however, in this case the phosphatase activity was partially suppressed, which could be an indication that Cys53 as a regulatory site, modulated by nitric oxide Intrigued by these findings, we decided to submit the protein variants (wild type, Dha53 mutant and C53A mutant) to incubation with H2O2 and nitric oxide. Surprisingly, both the chemical Dha53 and C53A mutants were extremely prone to H2O2 inactivation relative to the wild type and failed to be S-nitrosylated by NO. These findings have shown that the Dha-based posttranslational chemical mutagenesis facilitated the discovery of noncatalytic Cys53 as an oxidative-sensitive residue involved in a redox regulation mechanism; thus it functions as a scavenger of ROS and RNS, and reduces the overoxidation of catalytic Cys11. There is still the need to corroborate the role of Cys53 in the cellular activity and regulation of PtpA, and our results made its investigation in Mtb infection even more promising. The crystal structure of PtpA further showed that Cys53 was surface exposed and the solvent accessibility was the first answer for its preferential modification by α,α’-di-bromoadipyl(bis)amide compound (Figure 2). A prolonged protein incubation time with α,α’-di-bromoadipyl(bis)amide revealed an incomplete stoichiometric correlation in the chemical reactions, because it was not possible to modify all of the PtpA’s Cys residues completely under mild conditions.

Figure 2. Cys53 residue in M. tuberculosis PtpA and Cys259 residue in Y. enterocolitica YopH identified as the preferentially modified residues in our posttranslational chemical mutagenesis approach. Non-catalytic Cys53 is mostly exposed to the solvent in PtpA, whereas in YopH non-catalytic Cys259 is mostly buried. Their localization may account for their specific role, as PtpA Cys53 act as scavenger of reactive oxygen and nitrogen species and YopH Cys259 may act as a “backdoor” Cys as a result of its proximity to catalytic Cys403. Cartoons were generated with the CCP4 Molecular Graphics Program (University of York) with PDB files 1U2P (PtpA) and 4YAA (YopH).

In addition, failed attempts to chemically modify sitedirected double mutant C11/53A, which contains only Cys16 and the combined FTIR findings of disturbed O–H stretches in Dha11 and C11S mutants led us to identify non-catalytic Cys16 as a “backdoor” residue for our newly discovered Cys–Cys water-bridged motif (Figure 3). The elegant interplay between these non-catalytic residues (Cys16 and Cys53) shows how PtpA may prevent critical inhibition of its phosphatase activity under oxidative stress9. More fascinating is the fact that all this new information in regard to new structural motifs and regulation in PtpA may be used to design innovative antituberculosis (anti-PTPs) drugs. As a start, the design of Cys-directed electrophilic drug-like molecules that react preferentially with Cys53, which may disrupt its role as scavenger, by turning the protein more susceptible to oxidative damage or by inducing structural changes may lead to an inactive PtpA conformation. Targeting highly reactive non-catalytic Cys residues, such as Cys53, in PtpA with small molecules may avoid off-targets in drug development because it is not conserved amongst PTPs9. In fact, drug design strategies, like tethering and covalent fragment-based ligand discovery, can aid in the development of such small molecules67. These medicinal chemistry approaches have indeed led to the generation of FDA-approved drugs such as kinase-inhibitor ibrutinib and EGFR inhibitor afatinib, both of which targeti highly reactive non-catalytic Cys68, 69. New drugs could

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also be designed to target backdoor Cys16 and then disrupt the Cys–Cys water-bridged motif. Similar findings, that involve cryptic Cys residues, such as Cys333 in the PTP Shp2, support this argument70, which suggests that buried-yet-targetable allosteric sites can be used as scaffolds in computationally aided drug discovery. Because this new motif is also found in other bacterial phosphatases9, a drug design strategy that targets it could be an interesting alternative to fight resistant strains. Nevertheless, to avoid off-target effects parallel experiments must be performed to show if the Cys–Cys water-bridged motif is also present in the host’s phosphatases. CASE STUDY 2 – Non-catalytic Cys259 from Yersinina enterocolitica protein tyrosine phosphatase YopH YopH tyrosine phosphatase from Yersinia uses its catalytic Cys403 residue to hydrolyze a phosphotyrosine substrate, which usually involves the formation of a Cysphosphate intermediate. YopH contains a N-terminal signal, a chaperone binding region and a PTPase catalytic C-terminal domain, which is conserved among PTPs71. YopH Cys-phosphatase activity prevents invasintriggered bacteria internalization, by disrupting the focal adhesion complex12, 72 and suppresses oxidative burst in macrophages and neutrophils72. A combined inactivation of PRAM-1/SKAP-HOM and SLP76/Vav/PLCγ2 signal transduction axes and the modulation of lymphocytic activation ultimately mediates Yersinia survival73. Moreover, YopH contains another four Cys residues in addition to catalytic Cys403. Intriguingly, when YopH was submitted to the same posttranslational chemical reactions as PtpA it yielded once again preferential modification at non-catalytic Cys, except this time the preferentially modified Cys259 was located in a buried motif (Figure 2). A closer look at the crystal structure revealed that Cys259 is in proximity with Cys403, so these residues might interact with each other9. Cys259 in YopH may also act as “backdoor” Cys and play a similar role as Cys16 in PtpA. The presence of the Cys–Cys waterbridged motif in YopH remains to be confirmed. However, this new structural motif, which involves noncatalytic Cys units, is also present in SptpA9 from Staphylococcus aureus and is likely conserved within the LMW-PTP family (Figure 3). Our results clearly show that α,α’-di-bromoadipyl(bis)amide compound was able to distinguish residues based intrinsically in Cys reactivity, and that such reactivity is not necessarily determined by solvent accessibility, structural or folding constraints. It is, however, affected by a combination of all these features within the local environment and is highly protein-specific. This feature makes a Dha-based posttranslational chemical mutagenesis a robust methodology to generate point mutations in folded proteins and also to highlight intrinsic functionalities of its reactive Cys counterparts. Combined with site-directed mutagenesis a detailed overview of protein dynamics can be achieved and critical and conserved new binding motifs revealed.

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EXTENDING THE PLATFORM TO OTHER BACTERIAL PHOSPHATASES WITH MULTIPLE NONCATALYTIC CYS RESIDUES Bacterial phosphatases with multiple non-catalytic Cys residues are typically involved in polysaccharide production and as secreted virulence effectors, which downregulate the host-cell-signaling pathways74. SptP a tyrosine phosphatase from Salmonella typhimurium is another enzyme that relies on its Cys481 catalytic residue to circumvent host immune defenses11. It shares 30% sequence homology with YopH and is very similar structurally and enzymatically to this enzyme, except for the shape and charge distribution of its catalytic site75. SptP GAP activity inhibits signaling through Rho GTPases Rac-1 and Cdc42, which leads to rearrangements in the actin-cytoskeleton in host cells76. Similar to other secreted phosphatases, SptP suppresses the recruitment of neutrophils by dephosphorylating the vesicle fusion protein N-ethylamide-sensitive factor, which impedes the degranulation of local mast cells and facilitates bacterial spread77. Interestingly, SptP shares another structural feature with YopH, the presence of multiple noncatalytic Cys units, with unknown functions. TpbA tyrosine phosphatase from Pseudomonas aeruginosa is among the dual-specificity phosphatases that employ a catalytic Cys132 residue to dephosphorylate tyrosine, Ser and threonine residues in host proteins78. Pseudomonas successful infection is linked to its ability to form biofilms in a polymeric matrix79. Critically, TpbA reduces biofilm formation by the activity attenuation of TpbB, which leads to a reduced concentration of 3,5-cyclic diguanylic acid. This cyclic nucleotide is a second messenger that modulates the transcription of important genes involved in biofilm production and, consequently, important for the establishment of infection80. TbpA inactivation promotes rugose colony morphology, which is related to cell persistence in clinical infections80. TpbA also contains another two non-catalytic Cys residues spread along the amino acid sequence, which to date have no assigned function. Another example of multipleCys-containing bacterial phosphatase is the SP-PTP from Streptococcus pyogenes. SP-PTP possess an N-terminal signature motif C(X)5R(S/T) that is conserved in all LMW-PTPs81 and its catalytic function is linked to its Cys8 residue, which allows growth, cell division, cell adherence, and invasion of host cells. Once again, this phosphatase contains four other non-catalytic Cys residues distributed along its amino acid sequence82. These phosphatases represent good examples in which a Dhabased chemical mutagenesis approach may provide new relevant information on non-catalytic Cys function and interplay. This argument is strengthened by the fact that highly reactive non-catalytic Cys residues have already proven to be critical players in the survival of pathogenic bacteria under oxidative stress10.

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Biochemistry

Figure 3. Cys16 residue in M. tuberculosis PtpA identified as the “backdoor” Cys involved in the Cys11–Cys16 water-bridged motif by our posttranslational chemical mutagenesis approach. The motif is also found in S. aureus SptpA. It is likely that noncatalytic Cys13 may act as a “backdoor” Cys for SptpA in a mechanism similar to that identified for Cys16 in M. tuberculosis PtpA. Cartoons were generated with the CCP4 Molecular Graphics Program (University of York) with PDB files 1U2P (PtpA) and 3ROF (SptpA). Green numbers represent Cys8-Cys13 water bridged motif in S. aureus SptpA. Blue numbers represent Cys11-Cys16 water-bridged motif in M. tuberculosis PtpA.

CONCLUSION REMARKS & OUTLOOK Bacterial phosphatases play key roles and, in most cases, enable the successful survival of highly pathogenic bacteria inside human hosts, which represents a challenge for antibacterial therapeutics especially in multidrug-resistant bacteria. As shown in this perspective, a posttranslational chemical mutagenesis approach based on site-selective chemical modifications at Cys can be extended not only to produce diverse protein-conjugates, but also applied to reveal previously unknown amino acid functions in a biologically relevant context. As we have shown with the phosphatases, developing a drugoriented strategy that uses information provided by posttranslational mutagenesis may prove to be effective. The reactivity of the designed chemical probes and the residues of which they preferentially target can yet reveal unusual structural motifs and regulatory effects, which can then be target in an alternative and more direct drug design platform. The structural information may ultimately drive the synthesis of potent allosteric inhibitors. Eventually, by revealing important non-catalytic residues this platform can be extended to a vast set of proteins — not just enzymes — and structural proteins. This panel can include transcription factors, DNA and RNA binding proteins, receptors and chaperones. We have tested successfully the feasibility of the platform in a chaperone (in peer review process) and in an RNA binding protein (unpublished). Finally, as this robust chemical biology strategy develops over the next few years, it will afford a fast way to uncover the functionality of other key residues, such as lysines83, 84, aid in the answer of challenging biological questions and may ultimately mediate the development of a better and more specific drug-design platform.

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected] or [email protected] *E-mail: [email protected]

Funding Sources We thank CNPq Brazil (fellowship 200456/2015-6 to J.B.B., and grants 454507/2014-3 and 300606/2010-9 to H.T.), the European Regional Development Fund of the European Commission (EFRE) to (grant to S.H.), the Wilhelm-Roux Program (FKZ 31/09 to J.B.B.) and FCT Portugal (FCT Investigator to G.J.L.B.) for their support. We also thank Prof. Francisco Corzana for insightful discussions. G.J.L.B. is a Royal Society University Research Fellow and the recipient of a European Research Council Starting Grant (TagIt).

Notes The authors declare no conflict of interests. REFERENCES [1] Trifonov, E. N. (2004) The Triplet Code From First Principles, J. Biomol. Struct. Dyn. 22, 1-11. [2] Jordan, I. K., Kondrashov, F. A., Adzhubei, I. A., Wolf, Y. I., Koonin, E. V., Kondrashov, A. S., and Sunyaev, S. (2005) A universal trend of amino acid gain and loss in protein evolution, Nature 433, 633-638. [3] Barford, D. (2004) The role of Cys residues as redoxsensitive regulatory switches, Curr. Opin. Struct. Biol. 14, 679686. [4] Chalker, J.M., Bernardes, G.J.L., Lin, Y., and Davis, B.G. (2009) Chemical Modification of Proteins at Cys: Opportunities in Chemistry and Biology, Chem. Asian J. 4, 630-640. [5] Nagy, P., and Winterbourn, C. C. (2010) Chapter 6 - Redox Chemistry of Biological Thiols, In Advances in Molecular Toxicology (James, C. F., Ed.), pp 183-222, Elsevier. [6] Vogel, C., Silva, G. M., and Marcotte, E. M. (2011) Protein expression regulation under oxidative stress, Mol Cell Proteomics 10, M111 009217. [7] Rose, G. D., Geselowitz, A. R., Lesser, G. J., Lee, R. H., and Zehfus, M. H. (1985) Hydrophobicity of amino acid residues in globular proteins, Science 229, 834-838. [8] Marino, S. M., and Gladyshev, V. N. (2010) Cys Function Governs Its Conservation and Degeneration and Restricts Its Utilization on Protein Surfaces, J. Mol. Biol. 404, 902-916.

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