Drug Discovery is like a journey in which you dive into the biochemical space to fish out the compounds that fulfill the criteria of eventually becoming a drug candidate. Just like the outer space is full of stars that are usually grouped together into constellations, we can adopt the same concept in Drug Discovery. Here we consider that the Drug Discovery Stars are the Drug Targets for which ligands are diligently designed to modulate them. Usually, they are grouped into families (or constellations). We will address the common structural features about each family and discuss some important topics related to each one in a series of articles that we will be sharing here. We will start with Protein Kinases, one of the largest enzyme families in the human genome-encoded proteins – The Kinome.
Protein kinases functions and classifications:
Protein kinases have gained much attention in the Drug Discovery community as many of them are proved to be valid drug targets as they are being involved in the regulation and development of various diseases, the most famous of them is cancer, in addition to other important diseases, such as diabetes, inflammation, cardiovascular disorders, and infectious diseases. All kinases catalyze the transfer of a phosphate group from ATP to an acceptor hydroxyl – containing amino acid in the substrate protein usually serine, threonine, or tyrosine. Based on the type of the acceptor amino acid, kinases are categorized into the serine/threonine, and the tyrosine protein kinases. You may be wondering what the importance and implications of such protein phosphorylation are, the answer simply is that this phosphorylation modifies the activity of the protein through inducing conformational changes to be ready to mediate complex signal transduction cascades in the cell, hence regulating many essential functions such as transcription, mitosis, and cell proliferation .
Since All protein kinases catalyze the same phosphorylation reaction, they share the same structural fold, and have the same essential structural features. These structural features should be clearly understood in order to successfully design inhibitors. We will examine the crystal structure of cyclic adenosine monophosphate-dependent protein kinase (cAPK; protein kinase A) in complex with ATP and two manganese (II) ions, as a typical example for the protein kinase fold.
The main architecture:
There are two main subdomains in a kinase:
- The N-terminal lobe which is formed of a five-stranded β -sheet and one α -helix , called the a C helix [1, 2]. Inside this N- lobe a hydrophobic pocket is formed and it is where the adenine base of ATP binds. The positioning of this adenine base takes place through the formation of hydrogen bonds and Van der Waals forces with glycine residues which form the Glycine- rich loop -that will be discussed shortly- and the amide groups of the backbone .
- The larger C-terminal lobe which is mainly formed of α -helices. It binds the protein substrate[1, 2].
Those two lobes are connected through a flexible small chain (6-8 residues) called the hinge region.
The gatekeeper residue (Thr315), is an important residue that lies at the end of the hinge region in the N- terminal lobe. It has a crucial role in inhibitor binding and selectivity.
A closer look!
After knowing the general main architecture of kinases, it’s time to have a closer look on some important structural motifs of cAPK, as illustrated in Figure 1.
- A synthetic oligomeric peptide inhibitor, identifies the substrate-binding site.
- The glycine-rich loop or P-loop comprises the conserved motif GXGXXG and is mandatory for the phosphate residues positioning through hydrogen bonding. It functions as a lid, closing and opening the Nucleotide Binding Domain (NBD) to allow ATP to enter and ADP to leave.
- The α C helix has a glutamate residue which forms a salt bridge with a lysine residue in the β 3-strand of the N lobe. This Glu-Lys ion pair is essential for the catalytic function and is highly conserved in all protein kinases.
- The activation loop or A-loop contains hydroxyl-bearing amino acid residues that are phosphorylated to activate the enzyme to be able to perform its catalytic function and is conformationally highly flexible. This flexibility permits access to the NBD.
- Asp-Phe-Gly ( DFG-motif ) is an important conserved sequence among kinases, it is present in the magnesium-binding loop at the beginning of the A-loop in the N- terminal domain. Sometimes phenylalanine is replaced by leucine or tryptophan .
Figure 1. Structural features of protein kinases . Overall crystal structure of protein kinase A with bound ATP and Mn 2+. (PDB entry code 1ATP).
Carbon atoms are colored in grey, nitrogen atoms in blue, oxygen atoms in red, phosphor atoms in magenta and Sulfur atoms in yellow; hydrogen bonds are shown as dotted blue lines; and Mn 2+ ions are depicted as steel blue spheres. The residues 48 – 58 of the P-loop and residues 327 – 332 in the insert are omitted for clarity.
Active and inactive conformations- How it works!
- There is a signature sequence in protein kinases, that is K/E/D/D (Lys/Glu/Asp/Asp). The active conformation of all protein kinases features a salt bridge in the N-terminus (Lys72–Glu91). which matches the K/E part of K/E/D/D. Lys72 has a role in positioning the α- and β-phosphates of ATP as well. Another salt bridge is formed between Asp184 and the β- and γ-phosphates with the help of a magnesium ion. Asp184 corresponds to the D in the DFG (Asp-Phe-Gly) sequence. and it is also the first D of K/E/D/D.
- After binding of ATP and the protein substrate , the protein/peptide substrate hydroxyl- containing residue becomes close to the γ-phosphate of ATP. Asp166, which corresponds to the second D of K/E/D/D, acts as a general base and extracts the proton from the hydroxyl- containing residue.
- Lys168 neutralizes the negative charge of the γ-phosphate that is attacked by the oxygen on the hydroxyl- containing residue (e.g. serine) to form a metaphosphate intermediate (transition state) resulting in the release of ADP, and the formation of protein-serine phosphate.
- The peptide substrate has Arg-Arg-X-Z-Y sequence, where X is any amino acid, Z is a hydroxyl-containing amino acid, and Y is generally a hydrophobic residue .
Figure 2 The protein kinase reaction pathway . Asp166 acts as a general base and abstracts a proton from the substrate seryl group. A metaphosphate intermediate occurs in the transition state.
DFG-in and DFG-out:
One of the most important conformational changes affecting the state of activity of protein kinases is the conformation of the DFG motif. The DFG motif adopts two main conformations; the DFG-in conformation, where the phenylalanine side chain is buried inside the protein and oriented toward the α C helix rendering the protein in the active state, and the DFG-out conformation where the DFG residues flip of about 180ο, leading to changing the orientation of the phenylalanine side chain to be pointing outward to the surface of the protein kinase and consequently, access to the NBD is blocked, however a deep pocket is formed where inhibitors can bind. The movement of the DFG-motif covers approximately a 10 Å distance between the residues 
In order to illustrate the significant conformational changes between the active and the inactive states, Abl kinase was taken as an example, where its crystal structures binding with both ADP and imatinib (inhibitor) is shown in Figure 3.
Figure 3 Abl protein kinase in the active and inactive states . (a) Crystal structure of Abl kinase in complex with ADP (PDB code 2G2I), left, and in complex with imatinib (PDB code 1IEP), right. (b) Overlay of the ATP binding sites of Abl kinase in the active (green) and inactive (gray) conformation. The distance between the centers of both phenyl rings of Phe382 is 8.8 Å. The protein backbone is shown in gray ribbons. Both the activation loop (residues 381–409) and the ligand surface are colored in green for ADP and in blue for 1, respectively. The DFG-motif is colored in yellow, and the phenylalanine side chain of Phe382 is shown. The deep pocket accessible in the 1–Abl complex upon conformational rearrangement of the activation loop is emphasized in red.
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