Mycobacterium tuberculosis serine/threonine kinases PknB, PknD, PknE, and PknF phosphorylate multiple FHA domains

The physiologic roles and the substrates of the Mycobacterium tuberculosis (Mtb) serine/threonine kinases are largely unknown. Here, we report six novel interactions of PknB, PknD, PknE, and PknF with the Forkhead-Associated (FHA) domains of Rv0020c and the putative ABC transporter Rv1747. Purified PknB and PknF kinase domains phosphorylated multiple FHA-domain proteins in vitro. Although they remain to be verified in vivo, these reactions suggest a web of interactions between STPKs and FHA domains.     

Forkhead-associated Domain-containing Protein Rv0019c and Polyketide-associated Protein PapA5, from Substrates of Serine/Threonine Protein Kinase PknB to Interacting Proteins of Mycobacterium tuberculosis

Mycobacterium tuberculosis profoundly exploits protein phosphorylation events carried out by serine/threonine protein kinases (STPKs) for its survival and pathogenicity. Forkhead-associated domains (FHA), the phosphorylation-responsive modules, have emerged as prominent players in STPK mediated signaling. In this study, we demonstrate the association of the previously uncharacterized FHA domain-containing protein Rv0019c with cognate STPK PknB. The consequent phosphorylation of Rv0019c is shown to be dependent on the conserved residues in the Rv0019c FHA domain and activation loop of PknB. Furthermore, by creating deletion mutants we identify Thr³⁶ as the primary phosphorylation site in Rv0019c. During purification of Rv0019c from Escherichia coli, the E. coli protein chloramphenicol acetyltransferase (CAT) specifically and reproducibly copurifies with Rv0019c in a FHA domain-dependent manner. On the basis of structural similarity of E. coli CAT with M. tuberculosis PapA5, a protein involved in phthiocerol dimycocerosate biosynthesis, PapA5 is identified as an interaction partner of Rv0019c. The interaction studies on PapA5, purified as an unphosphorylated protein from E. coli, with Rv0019c deletion mutants reveal that the residues N-terminal to the functional FHA domain of Rv0019c are critical for formation of the Rv0019c-PapA5 complex and thus constitute a previously unidentified phosphoindependent binding motif. Finally, PapA5 is shown to be phosphorylated on threonine residue(s) by PknB, whereas serine/threonine phosphatase Mstp completely reverses the phosphorylation. Thus, our data provides initial clues for a possible regulation of PapA5 and hence the phthiocerol dimycocerosate biosynthesis by PknB, either by direct phosphorylation of PapA5 or indirectly through Rv0019c.     

Intramolecular regulatory switch in ZAP-70: analogy with receptor tyrosine kinases

ZAP-70, a Syk family cytoplasmic protein tyrosine kinase (PTK), is required to couple the activated T-cell antigen receptor (TCR) to downstream signaling pathways. It contains two tandem SH2 domains that bind to phosphorylated TCR subunits and a C-terminal catalytic domain. The region connecting the SH2 domains with the kinase domain, termed interdomain B, has previously been shown to have striking regulatory effects on ZAP-70 function, presumed to be due to the recruitment of key substrates. Paradoxically, deletion of interdomain B preserves ZAP-70 function. Recent structural studies of several receptor tyrosine kinases (RTKs) revealed that their juxtamembrane regions negatively regulate their catalytic activities. In EphB2 and several other RTKs, this autoinhibition depends upon interaction between the kinase domain and tyrosine residues within the juxtamembrane region. Autoinhibition is released when these tyrosines become phosphorylated following receptor stimulation. Sequence homology suggested analogous regulation for ZAP-70. Based on mutagenesis analysis of ZAP-70 interdomain B, we find that this region downregulates ZAP-70 catalytic activity in a similar manner as the juxtamembrane region of EphB2. Similar regulation was also noted for the related Syk kinase. These findings suggest that a general autoinhibitory mechanism employed by RTKs is also used by some cytoplasmic tyrosine kinases.     

Protein Kinase B (PknB) of Mycobacterium tuberculosis Is Essential for Growth of the Pathogen in Vitro as well as for Survival within the Host

The Mycobacterium tuberculosis protein kinase B (PknB) comprises an intracellular kinase domain, connected through a transmembrane domain to an extracellular region that contains four PASTA domains. The present study describes the comprehensive analysis of different domains of PknB in the context of viability in avirulent and virulent mycobacteria. We find stringent regulation of PknB expression necessary for cell survival, with depletion or overexpression of PknB leading to cell death. Although PknB-mediated kinase activity is essential for cell survival, active kinase lacking the transmembrane or extracellular domain fails to complement conditional mutants not expressing PknB. By creating chimeric kinases, we find that the intracellular kinase domain has unique functions in the virulent strain, which cannot be substituted by other kinases. Interestingly, we find that although the presence of the C-terminal PASTA domain is dispensable in the avirulent M. smegmatis, all four PASTA domains are essential in M. tuberculosis. The differential behavior of PknB vis-à-vis the number of essential PASTA domains and the specificity of kinase domain functions suggest that PknB-mediated growth and signaling events differ in virulent compared with avirulent mycobacteria. Mouse infection studies performed to determine the role of PknB in mediating pathogen survival in the host demonstrate that PknB is not only critical for growth of the pathogen in vitro but is also essential for the survival of the pathogen in the host.     

Conserved autophosphorylation pattern in activation loops and juxtamembrane regions of Mycobacterium tuberculosis Ser/Thr protein kinases

The identification of phosphorylation sites in proteins provides a powerful tool to study signal transduction pathways and to establish interaction networks involving signaling elements. Using different strategies to identify phosphorylated residues, we report here mass spectrometry studies of the entire intracellular regions of four 'receptor-like' protein kinases from Mycobacterium tuberculosis (PknB, PknD, PknE, and PknF), each consisting of an N-terminal kinase domain and a juxtamembrane region of varying length (26-100 residues). The enzymes were observed to incorporate different numbers of phosphates, from five in PknB up to 11 in PknD or PknE, and all detected sites were dephosphorylated by the cognate mycobacterial phosphatase PstP. Comparison of the phosphorylation patterns reveals two recurrent clusters of pThr/pSer residues, respectively, in their activation loops and juxtamembrane regions, which have a distinct effect on kinase activity. All studied kinases have at least two conserved phosphorylated residues in their activation loop and mutations of these residues in PknB significantly decreased the kinase activity, whereas deletion of the entire juxtamembrane regions in PknB and PknF had little effect on their activities. These results reinforce the hypothesis that mycobacterial kinase regulation includes a conserved activation loop mechanism, and suggest that phosphorylation sites in the juxtamembrane region might be involved in putative kinase-mediated signaling cascades.     

The extracytoplasmic domain of the Mycobacterium tuberculosis Ser/Thr kinase PknB binds specific muropeptides and is required for PknB localization

The Mycobacterium tuberculosis Ser/Thr kinase PknB has been implicated in the regulation of cell growth and morphology in this organism. The extracytoplasmic domain of this membrane protein comprises four penicillin binding protein and Ser/Thr kinase associated (PASTA) domains, which are predicted to bind stem peptides of peptidoglycan. Using a comprehensive library of synthetic muropeptides, we demonstrate that the extracytoplasmic domain of PknB binds muropeptides in a manner dependent on the presence of specific amino acids at the second and third positions of the stem peptide, and on the presence of the sugar moiety N-acetylmuramic acid linked to the peptide. We further show that PknB localizes strongly to the mid-cell and also to the cell poles, and that the extracytoplasmic domain is required for PknB localization. In contrast to strong growth stimulation by conditioned medium, we observe no growth stimulation of M. tuberculosis by a synthetic muropeptide with high affinity for the PknB PASTAs. We do find a moderate effect of a high affinity peptide on resuscitation of dormant cells. While the PASTA domains of PknB may play a role in stimulating growth by binding exogenous peptidoglycan fragments, our data indicate that a major function of these domains is for proper PknB localization, likely through binding of peptidoglycan fragments produced locally at the mid-cell and the cell poles. These data suggest a model in which PknB is targeted to the sites of peptidoglycan turnover to regulate cell growth and cell division.     

Phosphoprotein phosphatase of Mycobacterium tuberculosis dephosphorylates serine-threonine kinases PknA and PknB

The regulation of cellular processes by the modulation of protein phosphorylation/dephosphorylation is fundamental to a large number of processes in living organisms. These processes are carried out by specific protein kinases and phosphatases. In this study, a previously uncharacterized gene (Rv0018c) of Mycobacterium tuberculosis, designated as mycobacterial Ser/Thr phosphatase (mstp), was cloned, expressed in Escherichia coli, and purified as a histidine-tagged protein. Purified protein (Mstp) dephosphorylated the phosphorylated Ser/Thr residues of myelin basic protein (MBP), histone, and casein but failed to dephosphorylate phospho-tyrosine residue of these substrates, suggesting that this phosphatase is specific for Ser/Thr residues. It has been suggested that mstp is a part of a gene cluster that also includes two Ser/Thr kinases pknA and pknB. We show that Mstp is a trans-membrane protein that dephosphorylates phosphorylated PknA and PknB. Southern blot analysis revealed that mstp is absent in the fast growing saprophytes Mycobacterium smegmatis and Mycobacterium fortuitum. PknA has been shown, whereas PknB has been proposed to play a role in cell division. The presence of mstp in slow growing mycobacterial species, its trans-membrane localization, and ability to dephosphorylate phosphorylated PknA and PknB implicates that Mstp may play a role in regulating cell division in M. tuberculosis.     

Proteomic identification of M. tuberculosis protein kinase substrates: PknB recruits GarA, a FHA domain-containing protein, through activation loop-mediated interactions

Genes for functional Ser/Thr protein kinases (STPKs) are ubiquitous in prokaryotic genomes, but little is known about their physiological substrates and their actual involvement in bacterial signal transduction pathways. We report here the identification of GarA (Rv1827), a Forkhead-associated (FHA) domain-containing protein, as a putative physiological substrate of PknB, an essential Ser/Thr protein kinase from Mycobacterium tuberculosis. Using a global proteomic approach, GarA was found to be the best detectable substrate of the PknB catalytic domain in non-denatured whole-cell protein extracts from M. tuberculosis and the saprophyte Mycobacterium smegmatis. Enzymological and binding studies of the recombinant proteins demonstrate that docking interactions between the activation loop of PknB and the C-terminal FHA domain of GarA are required to enable efficient phosphorylation at a single N-terminal threonine residue, Thr22, of the substrate. The predicted amino acid sequence of the garA gene, including both the N-terminal phosphorylation motif and the FHA domain, is strongly conserved in mycobacteria and other related actinomycetes, suggesting a functional role of GarA in putative STPK-mediated signal transduction pathways. The ensuing model of PknB-GarA interactions suggests a substrate recruitment mechanism that might apply to other mycobacterial kinases bearing multiple phosphorylation sites in their activation loops.     

The effect of HAMP domains on class IIIb adenylyl cyclases from Mycobacterium tuberculosis.

The genes Rv1318c, Rv1319c, Rv1320c and Rv3645 of Mycobacterium tuberculosis are predicted to code for four out of 15 adenylyl cyclases in this pathogen. The proteins consist of a membrane anchor, a HAMP region and a class IIIb adenylyl cyclase catalytic domain. Expression and purification of the isolated catalytic domains yielded adenylyl cyclase activity for all four recombinant proteins. Expression of the HAMP region fused to the catalytic domain increased activity in Rv3645 21-fold and slightly reduced activity in Rv1319c by 70%, demonstrating isoform-specific effects of the HAMP domains. Point mutations were generated to remove predicted hydrophobic protein surfaces in the HAMP domains. The mutations further stimulated activity in Rv3645 eight-fold, whereas the effect on Rv1319c was marginal. Thus HAMP domains can act directly as modulators of adenylyl cyclase activity. The modulatory properties of the HAMP domains were confirmed by swapping them between Rv1319c and Rv3645. The data indicate that in the mycobacterial adenylyl cyclases the HAMP domains do not display a uniform regulatory input but instead each form a distinct signaling unit with its adjoining catalytic domain.     

Protein Kinase A (PknA) of Mycobacterium tuberculosis Is Independently Activated and Is Critical for Growth in Vitro and Survival of the Pathogen in the Host

The essential mycobacterial protein kinases PknA and PknB play crucial roles in modulating cell shape and division. However, the precise in vivo functional aspects of PknA have not been investigated. This study aims to dissect the role of PknA in mediating cell survival in vitro as well as in vivo. We observed aberrant cell shape and severe growth defects when PknA was depleted. Using the mouse infection model, we observe that PknA is essential for survival of the pathogen in the host. Complementation studies affirm the importance of the kinase, juxtamembrane, and transmembrane domains of PknA. Surprisingly, the extracytoplasmic domain is dispensable for cell growth and survival in vitro. We find that phosphorylation of the activation loop at Thr¹⁷² of PknA is critical for bacterial growth. PknB has been previously suggested to be the receptor kinase, which activates multiple kinases, including PknA, by trans-phosphorylating their activation loop residues. Using phospho-specific PknA antibodies and conditional pknB mutant, we find that PknA autophosphorylates its activation loop independent of PknB. Fluorescently tagged PknA and PknB show distinctive distribution patterns within the cell, suggesting that although both kinases are known to modulate cell shape and division, their modes of action are likely to be different. This is supported by our findings that expression of kinase-dead PknA versus kinase-dead PknB in mycobacterial cells leads to different cellular phenotypes. Data indicate that although PknA and PknB are expressed as part of the same operon, they appear to be regulating cellular processes through divergent signaling pathways.     

Structure of Mycobacterium tuberculosis PknB supports a universal activation mechanism for Ser/Thr protein kinases

A family of eukaryotic-like Ser/Thr protein kinases occurs in bacteria, but little is known about the structures and functions of these proteins. Here we characterize PknB, a transmembrane signaling kinase from Mycobacterium tuberculosis. The intracellular PknB kinase domain is active autonomously, and the active enzyme is phosphorylated on residues homologous to regulatory phospho-acceptors in eukaryotic Ser/Thr kinases. The crystal structure of the PknB kinase domain in complex with an ATP analog reveals the active conformation. The predicted fold of the PknB extracellular domain matches the proposed targeting domain of penicillin-binding protein 2x. The structural and chemical similarities of PknB to metazoan homologs support a universal activation mechanism of Ser/Thr protein kinases in prokaryotes and eukaryotes.     

The Ser/Thr protein kinase PknB is essential for sustaining mycobacterial growth

The receptor-like protein kinase PknB from Mycobacterium tuberculosis is encoded by the distal gene in a highly conserved operon, present in all actinobacteria, that may control cell shape and cell division. Genes coding for a PknB-like protein kinase are also found in many more distantly related gram-positive bacteria. Here, we report that the pknB gene can be disrupted by allelic replacement in M. tuberculosis and the saprophyte Mycobacterium smegmatis only in the presence of a second functional copy of the gene. We also demonstrate that eukaryotic Ser/Thr protein kinase inhibitors, which inactivate PknB in vitro with a 50% inhibitory concentration in the submicromolar range, are able to kill M. tuberculosis H37Rv, M. smegmatis mc(2)155, and Mycobacterium aurum A+ with MICs in the micromolar range. Furthermore, significantly higher concentrations of these compounds are required to inhibit growth of M. smegmatis strains overexpressing PknB, suggesting that this protein kinase is the molecular target. These findings demonstrate that the Ser/Thr protein kinase PknB is essential for sustaining mycobacterial growth and support the development of protein kinase inhibitors as new potential antituberculosis drugs.     

Complex effects of naturally occurring mutations in the JAK3 pseudokinase domain: evidence for interactions between the kinase and pseudokinase domains

The structure of Janus kinases (JAKs) is unique among protein tyrosine kinases in having tandem, nonidentical kinase and pseudokinase domains. Despite its conservation in evolution, however, the function of the pseudokinase domain remains poorly understood. Lack of JAK3 expression results in severe combined immunodeficiency (SCID). In this study, we analyze two SCID patients with mutations in the JAK3 pseudokinase domain, which allows for protein expression but disrupts the regulation of the kinase activity. Specifically, these mutant forms of JAK3 had undetectable kinase activity in vitro but were hyperphosphorylated both in patients' Epstein-Barr virus-transformed B cells and when overexpressed in COS7 cells. Moreover, reconstitution of cells with these mutants demonstrated that, although they were constitutively phosphorylated basally, they were unable to transmit cytokine-dependent signals. Further analysis showed that the isolated catalytic domain of JAK3 was functional whereas either the addition of the pseudokinase domain or its deletion from the full-length molecule reduced catalytic activity. Through coimmunoprecipitation of the isolated pseudokinase domain with the isolated catalytic domain, we provide the first evidence that these two domains interact. Furthermore, whereas the wild-type pseudokinase domain modestly inhibited kinase domain-mediated STAT5 phosphorylation, the patient-derived mutants markedly inhibited this phosphorylation. We thus conclude that the JAK3 pseudokinase domain is essential for JAK3 function by regulating its catalytic activity and autophosphorylation. We propose a model in which this occurs via intramolecular interaction with the kinase domain and that increased inhibition of kinase activity by the pseudokinase domain likely contributes to the disease pathogenesis in these two patients.     

Interaction of Rv1625c, a mycobacterial class IIIa adenylyl cyclase, with a mammalian congener

The adenylyl cyclase Rv1625c from Mycobacterium tuberculosis codes for a protein with six transmembrane spans and a catalytic domain, i.e. it corresponds to one half of the pseudoheterodimeric mammalian adenylyl cyclases (ACs). Rv1625c is active as a homodimer. We investigated the role of the Rv1625c membrane domain and demonstrate that it efficiently dimerizes the protein resulting in a 7.5-fold drop in K(m) for ATP. Next, we generated a duplicated Rv1625c AC dimer by a head-to-tail concatenation. This produced an AC with a domain order exactly as the mammalian pseudoheterodimers. It displayed positive cooperativity and a 60% increase of v(max) compared with the Rv1625c monomer. Further, we probed the compatibility of mycobacterial and mammalian membrane domains. The second membrane anchor in the Rv1625c concatamer was replaced with membrane domain I or II of rabbit type V AC. The mycobacterial and either mammalian membrane domains are compatible with each other and both recombinant proteins are active. A M. tuberculosis Rv1625c knockout strain was assayed in a mouse infection model. In vitro growth characteristics and in vivo organ infection and mortality were unaltered in the knockout strain indicating that AC Rv1625c alone is not a virulence factor.     

Protein kinase C is involved in the regulation of several calreticulin posttranslational modifications

Calreticulin (CRT) is a highly versatile lectin-like chaperone that affects many cellular functions both inside and outside the endoplasmic reticulum lumen. We previously reported that calreticulin interacts with several protein kinase C isozymes both in vitro and in vivo. The aim of this study was to elucidate the molecular determinants involved in the association between these proteins and the biochemical significance of their interaction. Using full-length or CRT-domain constructs expressed as GST-fusion proteins, we found that protein kinase C binds to the CRT N domain in overlay and pull-down assays. Phosphorylation experiments showed that only this CRT domain is phosphorylated by the kinase. Lectin blot analysis demonstrated that CRT is modified by N-glycosylation, but this modification did not affect its interaction with protein kinase C. We also demonstrated that although both domains of protein kinase C theta can bind to CRT, it is the catalytic one that binds with higher affinity to CRT. Immunofluorescence studies showed that CRT and PKC co-localize mainly at the ER (estimated in 35%). Activation of protein kinase C induced caused transient changes in CRT localization, and unexpectedly, also induced changes in posttranslational modifications found in the protein: CRT N-glycosylation is abolished, whereas tyrosine phosphorylation and O-linked β-N-acetylglucosamine modification are increased. Together, these findings suggest that protein kinase C is involved in the regulation of CRT function.     

The Mycobacterium tuberculosis serine/threonine kinase PknL phosphorylates Rv2175c: mass spectrometric profiling of the activation loop phosphorylation sites and their role in the recruitment of Rv2175c

Although Mycobacterium tuberculosis (M. tb) comprises 11 serine/threonine protein kinases, the mechanisms of regulation of these kinases and the nature of their endogenous substrates remain largely unknown. Herein, we characterized the M. tb kinase PknL by demonstrating that it expresses autophosphorylation activity and phosphorylates Rv2175c. On-target dephosphorylation/MALDI-TOF for identification of phosphorylated peptides was used in combination with LC-ESI/MS/MS for localization of phosphorylation sites. By doing so, five phosphorylated threonine residues were identified in PknL. Among them, we showed that the activation loop phosphorylated residues Thr173 and Thr175 were essential for the autophosphorylation activity of PknL. Phosphorylation of the activation loop Thr173 residue is also required for optimal PknL-mediated phosphorylation of Rv2175c. Together, our results indicate that phosphorylation of the PknL activation loop Thr residues not only controls PknL kinase activity but is also required for recruitment and phosphorylation of its substrate. Rv2175c was found to be phosphorylated when overexpressed and purified from Mycobacterium smegmatis as 2-DE indicated the presence of different phosphorylated isoforms. Given the presence of the dcw gene cluster in the close vicinity of the pknL/Rv2175c locus, and its conservation in all mycobacterial species, we propose that PknL/Rv2175c may represent a functional pair in the regulation of mycobacterial cell division and cell envelope biosynthesis.     

Interdomain interaction reconstitutes the functionality of PknA, a eukaryotic type Ser/Thr kinase from Mycobacterium tuberculosis

Eukaryotic type Ser/Thr protein kinases have recently been shown to regulate a variety of cellular functions in bacteria. PknA, a transmembrane Ser/Thr protein kinase from Mycobacterium tuberculosis, when constitutively expressed in Escherichia coli resulted in cell elongation and therefore has been thought to be regulating morphological changes associated with cell division. Bioinformatic analysis revealed that PknA has N-terminal catalytic, juxtamembrane, transmembrane, and C-terminal extracellular domains, like known eukaryotic type Ser/Thr protein kinases from other bacteria. To identify the minimum region capable of exhibiting phosphorylation activity of PknA, we created several deletion mutants. Surprisingly, we found that the catalytic domain itself was not sufficient for exhibiting phosphorylation ability of PknA. However, the juxtamembrane region together with the kinase domain was necessary for the enzymatic activity and thus constitutes the catalytic core of PknA. Utilizing this core, we deduce that the autophosphorylation of PknA is an intermolecular event. Interestingly, the core itself was unable to restore the cell elongation phenotype as manifested by the full-length protein in E. coli; however, its co-expression along with the C-terminal region of PknA can associate them in trans to reconstitute a functional protein in vivo. Therefore, these findings argue that the transmembrane and extracellular domains of PknA, although dispensable for phosphorylation activities, are crucial in responding to signals. Thus, our results for the first time establish the significance of different domains in a bacterial eukaryotic type Ser/Thr kinase for reconstitution of its functionality.     

The juxtamembrane region of MuSK has a critical role in agrin-mediated signaling

MuSK is a receptor tyrosine kinase expressed selectively in skeletal muscle and localized to neuromuscular synapses. Agrin activates MuSK and stimulates phosphorylation and clustering of acetylcholine receptors (AChRs) at synaptic sites. We expressed wild-type or mutant MuSK in MuSK(-/-) myotubes and identified tyrosine residues in the MuSK cytoplasmic domain that are necessary for agrin-stimulated phosphorylation and clustering of AChRs. The activation loop tyrosines and the single juxtamembrane tyrosine were found to be essential for agrin-stimulated phosphorylation and clustering of AChRs. Further, we show that the juxtamembrane tyrosine, contained within an NPXY motif, is phosphorylated in vivo by agrin stimulation. We constructed chimeras containing extracellular and transmembrane domains from MuSK and cytoplasmic sequences from TrkA and found that inclusion of 13 amino acids from the MuSK juxtamembrane region, including the NPXY motif, is sufficient to convert a phosphorylated but inactive MuSK-TrkA chimera into a phosphorylated active chimera. These data suggest that phosphorylation of the MuSK NPXY site leads to recruitment of a phosphotyrosine-binding domain-containing protein that functions to stimulate phosphorylation and clustering of AChRs.     

Structural and functional analysis of phosphothreonine-dependent FHA domain interactions

FHA domains are well established as phospho-dependent binding modules mediating signal transduction in Ser/Thr kinase signaling networks in both eukaryotic and prokaryotic species. Although they are unique in binding exclusively to phosphothreonine, the basis for this discrimination over phosphoserine has remained elusive. Here, we attempt to dissect overall binding specificity at the molecular level. We first determined the optimal peptide sequence for Rv0020c FHA domain binding by oriented peptide library screening. This served as a basis for systematic mutagenic and binding analyses, allowing us to derive relative thermodynamic contributions of conserved protein and peptide residues to binding and specificity. Structures of phosphopeptide-bound and uncomplexed Rv0020c FHA domain then directed molecular dynamics simulations which show how the extraordinary discrimination in favor of phosphothreonine occurs through formation of additional hydrogen-bonding networks that are ultimately stabilized by van der Waals interactions of the phosphothreonine γ-methyl group with a conserved pocket on the FHA domain surface.