What determinant residues?

In the example protein kinase sequence, the (semi)conserved residues/motifs are colored in red. Determinant residues are located in relation to these.

e.g. AMK+11=R, GEL+1=E, DFG+3=F, APE-1=L, etc.

There are as many as 20 determinates in a protein kinase.

Here is a list of the determinants.

Determinant # - Motif Sequence - (Sub-site)

List of Determinants (in sequence order). Most can have sequence variability.
Sub-domain I:
1 GXGXXG (-1) (Gly-rich loop) Van der Waals interactions with Leu, Phe etc.
2 GXGXXG (-1) or (+2) Weak interaction. Commonly Ala, Ser or Thr. Gly in many Arabidopsis or C. elegans protein kinases inter alia (GXGGXG).
3 GXGXXG (+2) Alternative, or complementary, site to determinants 4 and 5. Commonly Phe or Tyr.
GXG in S. cerevisiae CAK1 appears to be at the C-terminal rather than at the N-terminal (304 - 306) which suggests an unusual folding pattern.
Sub-domain II:
*ALK (AMK, AVK, AIK, VVK etc.) motif almost always present (but not found in WNK's, edit AWC to AVK). Usually about 20 residues downstream of GXG.
NOTE: This is the Lys which is mutated to Arg to create a "dead" kinase.
4 In the loop at the N-terminal end of the C-helix (+2) Not used in CDK's (where these residues are involved in cyclin binding) or PHK (where the (+2) Arg binds to determinant 12 instead).
Sub-domain III:
5 In the loop at the N-terminal end of the C-helix.(+2)
Conserved *Glu in the C-helix. The number of residues between the *Lys and *Glu residues determines the location of determinants 4 and 5. For *Glu = *Lys+19, as in PKA and PKC, 4 and 5 are at *Lys+10 and *Lys + 12, as in A MKILDKQKVV KLKQIEHTLN E (PKA) or AIKILKKDVVIQDDDVECTMVE (PKCa). Determinants 4 and 5 are difficult to determine if the K/E loop is longer than 20 residues, with *Glu-6 or -7 being the best guide.
Sub-domain IV:
GELXXX The hinge or linker region. The three underlined residues are:
(1) Gly is the normal residue in AGC/CaMK protein kinases, but it can be replaced by Ser, Asn or Lys in other groups,
(2) 6 Glu (-3) (Arg-specific) can be replaced by Asp, Ser, Ala or Pro; and
(3) Leu (by far the most common residue in this position) It can be replaced by Met, Val or Il.e. PKA has Met in this position (GEMFSH).
7 GELXXX (-3) Mostly hydrophobic. PKA has Phe at 7. The side-chains of some PKA and PKC substrates with Leu, Pro or Met at (-3), bind to this residue. Becomes more important when there is a small residue, such as Ser, are at determinant 6.
8 GELXXX (-3) or (-4) Minor possibility. Very variable.
This motif can be identified by the residues immediately upstream. A Glu framed by two hydrophobics e.g. MEY, is followed by another hydrophobic residue. This is then followed by two hydrophilic or bend-forming residues, the second of which is often Gly. This residue is missing in CDK, MAPK and GSK3b kinases. The Gly is followed by the GELXXX motif. The full sequence is in PKA is: MEYLEGGEMFSH.
:Sub-domain VIb:
9 HRDLKPEN Determinant 9 is the catalytic Asp. Also noted is the Mg-binding Asn. Both residues must be present, and are found in all protein kinase families, including tyrosine protein kinases and PI3K-like kinases such as ATM. Atypically, the Asn is replaced by Thr in Xenopus laevis BUB1. Usually about 100 residues downstream of ALK. The His in HRD is a Tyr in most AGC protein kinases.
10 HRDLKPEN (-2) or (-3). (-3) in Pro-directed kinases (CDK's and MAPK's). Glu can be replaced by Ser, Gln, Gly, Asn etc. PEN is ADN in PKC isozymes.
Sub-domain VII:
11 DFGL (+1) or (+2) Usually hydrophobic, at the back of the (+1) pocket. Minor possibility. The binding site for the (+1) Pro in CDK's and MAPK's.
NOTE: The highly conserved "D" in the DFG motif has an important role in the enzymatic activity of protein kinases as a ligand of one of the Mg ions. It is not, however, the major catalytic Asp involved in phosphate transfer, which instead is the Asp of HRDLKPEN. This was shown in the crystal structures of PKA and PHK in the 1990s.
Sub-domain VIII:
12 The activation loop Thr or Ser (or Glu in PHK) which is nearly always phosphorylated. (+3) (minor possibility). RTWTLCG in PKA or TYTHEVV in CDK2. Located nine residues upstream of APE in CaMK and AGC kinases; ten residues upstream of APE in CDK's and MAPK's. Some protein kinases have no activation loop pThr or pSer. Notable examples include PHK, Chk1 and CK2.
13 The residue immediately following determinant 12 (at the end of the activation loop). (+3) or (+1). On the side of the (+1) pocket. RTWTLCG in PKA.
CGTPEYLAPE No contacts with substrate side-chains, but the peptide backbone does form an anti-parallel b-sheet with the peptide backbone of the (+1) and (+2) residues of the substrate in AGC and CaMK protein kinases. Cys can often be replaced by Val.
14 CGTPEYLAPE (-2) Often Thr, but sometimes Ser. The Thr -CH3 binds to other residues with a b-methyl such as Ile, Val or Thr. This residue is usually not phosphorylated. The -OH points towards the catalytic Asp [Johnson LN, Lowe ED, Noble ME and Owen DJ. (1998) The Eleventh Datta Lecture. The structural basis for substrate recognition and control by protein kinases.FEBS Lett. 430, 1-11].
The seven residues preceding the APE motif are group-specific, and are not part of the activation loop. This is cxalled "the P+1 loop". The APE motif is usually conserved and readily identifiable, but it can be replaced by SPE, PPE, PIS, PLS, SIN, SID etc. Not easy to find in such protein kinases as CAK1 or BUB1.
15 CGTPEYLAPE (+1) On the side of the (+1) pocket. Usually Pro, residues such as Leu will partially occlude the (+1) pocket. Arg at this position (e.g. CK2) will spill over to the (-2) pocket, giving rise to a Glu specificity at (-2) and (+1). Less likely to be Pro in S. cerevisiae protein kinases than their equivalent human protein kinases.
16 CGTPEYLAPE (-2) Glu in AGC kinases (PKA, PKC, PKG etc.) where it is Arg or Lys-specific. Variable in CaMK family (Gly, Ser, Asn, Cys etc.) and determines (-2) specificity.
Protein kinases in the CaMK sub-group usually do not have a Glu at 16, except Rck1 from S. cerevisiae. 16 is Gly in CaMKII and this results in a very broad specificity.
Uniquely 16 is a His in Hsl1, Kcc4 and Gin4 in S. cerevisiae and may result in a specificity for Glu or Asp residuers.
In CDK's and MAPK's, a large aromatic residue, often Trp, partially blocks the (-2) pocket, so that the specificity at (-2) is for Pro, Val, Ala, Thr etc.
Human Chk1 has a "reversal" with Leu at 15 and a Pro at 16.
17 CGTPEYLAPE (-2) Almost invariably an aromatic residue (Tyr, Phe, Trp). The major binding site for the residue at (-2), unless it is small (such as Ser) and is modified by determinant 16. Note that a residue binding to Tyr can bind either to the phenyl ring or to the phenolic group.
Unless 16 is large (Trp), an Arg cabn still bind to a 17 Tyr in most cases.
The P+1 Loop
18 CGTPEYLAPE (+1) Often hydrophobic, at the bottom of the (+1) pocket. A phosphorylated Tyr in Titin, which binds an Arg at (+1).
Sub-domain IX:
19 WWALGVLIYE 221-230 of PKA (-2) WWGLGVVM YE 333-342 of AKT (-5). Arg-specific. Restricted to the AGC kinases. Not used in PREDIKIN. The competition between the (-2) and (-5) sites of Akt is instructive. [Yang J, Cron P, Thompson V, Good VM, Hess D, Hemmings BA and Barford D. (2002) Molecular mechanism for the regulation of protein kinase B/Akt by hydrophobic motif phosphorylation. Mol Cell 9,1227-40].
20 NFDDY 326-330 of PKA (-3) is Arg-specific. PKA specific.Not used in PREDIKIN..