PknB
Created by Chelsea Haakenson
Protein kinases are common regulatory enzymes found primarily in eukaryotes.1 They are the third most common protein domain in the human genome and make up two percent of eukaryotic proteins.2 A prototypical example is the Ser/Thr protein kinase of Mycobacterium tuberculosis, PknB. Scientists postulate that PknB is a potential regulator of cell growth and division as a consequence of its containing an operon including genes (rodA, pknA, and pbpA) important for that purpose.1 When PknB is inhibited, significant inhibitory effects of mycobacterial growth are observed, particularly when inhibition is caused by structures, such as K-252-a and K-252-b, that target the ATP-binding site.3 The change in cell morphology due to depletion or over expression of PknB and its predominant expression during exponential growth serve as other indicators of PknB’s involvement in cell growth .3
PknB is encoded by the gene Rv0014c and is a 626 amino acid trans-membrane protein with an intracellular N-terminal kinase domain and an extracellular C-terminal domain.4 It is a prototypical Ser/Thr protein kinase and contains a kinase homology domain (residues 11 – 268) followed by a 50 residue linker to a single transmembrane helix and a 274 residue extracellular domain composed of four PASTA domains.1 The PknB catalytic domain is composed of alpha and beta chains, has a total molecular weight of 33518.9, and has an isoelectric point of 5.0801.5 PknB has two ligands: a phosphothiophosphoric acid-adenylate ester (also known as ATP g-S), and a Mn2+ or Mg2+ divalent cation.5 Since the intracellular domain is active autonomously, discussion of PknB in this document is limited to that domain.
General Ser/Thr protein kinase structure is comprised of three principle parts: a region of hydrophobic residues clustered around the adenosine of ATP, an area around the g-phosphate of ATP enclosed primarily by charged residues (the active site), and a region in the large lobe situated below ATP composed of both hydrophobic and polar residues.6 PknB contains these three parts, with the small N-terminal lobe above ATP and the large C-terminal lobe below ATP giving the catalytic core a ‘bean-like’ structure.2
Some important structural elements of the PknB kinase domain include helix C, the P-loop, the catalytic loop, and the activation loop.1 Helix C is found in the linker segment joining the kinase homology domain to the transmembrane helix and its position may be controlled by the strict conservation of the intermolecular ion pair between Arg10 and Asp76, which makes intermolecular hydrophobic contact with Leu33 and an intramolecular hydrogen bond with Tyr11.1 The intramolecular contact with Leu33 connects the C-terminus of helix C to the ‘backside’ N-terminal lobe. Helix C also contains a highly conserved Glu59 which makes contact with Lys40.1 The hexapeptide 18-GFGGMS-23 forms the P-loop of PknB, in which the main chain amides of the glycines coordinate phosphates.1 The catalytic domain is composed of residues 1–279 and includes the ATP binding site.7 Residues 137–143 comprise the activation loop, which functions to coordinate the Mg2+ or Mn2+ ion through the contact between Asp156 and Asn143 to Asp138.1 The activation loop contains four of the six phosphorylated residues in PknB (Ser166, Ser173, Thr171, and Thr173 are found in the loop while Thr294 and Ser295 are not).1 Two of these residues, Thr171 and Thr173, were identified as the only sites that are fully autophosphorylated.7 Several distinct structural features, such as a tilt of the aC helix away from the active site, the conserved Glu59 in the aC helix, and disorder of the activation group, are present in PknB because the kinase domain crystallizes as a homodimer.8
Activation of PknB occurs when ATP binds between the small N-terminal lobe and large C-terminal lobe, directing the g-phosphate outwards while the adenine ring lies deep in the cleft between the two lobes.2 The residues in the large lobe serve to stabilize the region while the hydrophobic region around the adenosine creates a binding pocket for ATP and the charged residues in the active site bind and position the g-phosphate.6 In order to correctly position ATP, stabilize the active conformation, and facilitate the catalytic mechanism, five specific amino acids (Glu12, Lys9, Asp30, Asn33, and Asp36) are fully conserved in all the kinases.6 Lys9 interacts with the a- and b-phosphates of ATP to provide stabilization. Further stabilization is provided by the formation of a salt bridge between Lys9 and Glu12.6 Asp30 serves as the catalytic base to initiate phototransfer by deprotonating an acceptor serine, threonine, or tyrosine.6 Asn33 and Asp 36 interact with the divalent cation, indirectly positioning the g-phosphate of ATP.6
Ser/Thr protein kinases have several conserved catalytic residues (Lys40, Glu59, Asp138, and Asn143) important for catalyzing phosophoryl transfer.8 One protein that has been shown to be phosphorylated by PknB is PBPA, a penicillin-binding protein required for cell division.4 When PknB and PBPA are coexpressed, phosporylation of PBPA occurs on Thr437 and Thr362.4 Also implicated as a physiological substrate of PknB is the Forkhead-associated (FHA) domain-containing protein, GarA.7 Experimental results indicated that Thr22 of GarA is the phosphate-acceptor.7 This interaction is not surprising, as FHA domains are known to specifically bind phosphorylated threonine residues and the catalytic domain of PknB has four phosphorylated residues in its activation loop, with two of them (Thr171 and Thr173) fully phosphorylated.7 The existence of multiple phosphorylation sites in the kinase activation loop appears to be important for such intermolecular interactions because phosphate groups are required to bind both the FHA domain for substrate recruitment and the conserved Arg cluster (including Arg137) from the catalytic loop for enzyme activation.7
The function of PknB in phosphorylating certain proteins can be inhibited by two mechanisms: dephosphorylation and disruption of ATP binding. The protein phosphatase, PstP, from Mycobacterium tuberculosis serves as a regulator of PknB via dephosphorylation targeted at Thr171 and Thr173.9 This is an effective method of decreasing the functionality of PknB, since experimentally replacing these threonine residues with alanine significantly reduces kinase activity.9 Synthetic inhibitors often use a different method and are typically designed to block ATP from binding.6 This is also an effective method, since K-252-a and K-252-b, which contain the indole carbazole chromophore and target the ATP-binding site, produce significant inhibitory effects of PknB.3
One homologue of PknB is another Ser/Thr protein kinase, PknE. PknE is found in Mycobacterium tuberculosis, which encodes a total eleven of these kinases.7 It contains alpha and beta chains and has a molecular weight of 68242.68.10 The primary similarity between PknB and PknE is the three-dimensional structure and mechanism of activation and substrate recognition, features that are generally conserved among Ser/Thr protein kinases.7 The specific structural features that were conserved between PknB and PknE include the N and C-terminal lobes, the P loop (which binds the phosphate), and the catalytic loop.7 The most important shared feature of Ser/Thr protein kinases, however, is the conservation of five residues involved in catalyzing phosphoryl transfer (Lys45, Glu64, Asp157, Asp139, and Asn144 – numbering from PknE).7 The conservation of these sites is probably due to their importance in the general function of Ser/Thr protein kinases, in particular activation and phosphorylation. PknE does diverge considerably from PknB, however, a possible explanation for why PknE is not essential for bacterial growth.7 The function of specific differences cannot currently be explained though, because the exact role of PknE is not known.7
PknB serves an important role in cell growth and division and therefore needs a mechanism that has high sensitivity but maintains specificity. The arrangement of PknB into the helix C, the P-loop, the catalytic loop, and the activation loop helps to accomplish this goal.1 The use of phosphorylated serines and threonines as a catalytic mechanism allows for high sensitivity as there are many structures with residues available for phosphorylation. Binding of ATP between the small N-terminal lobe and the large C-terminal lobe, directing the g-phosphate outwards while the adenine ring lies deep in the hydrophobic cleft between the two lobes helps maintain specificity and stabilize activation.2 The need for multiple phosphorylation sites in the kinase activation loop also increases substrate specificity.7 Due to these features, the PknB structure satisfies its functional needs and allows for efficient biological activity.