Biochem assignment 2

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Protein Kinases

Structure and Regulation:

1)  Protein Kinase A (Lehninger Ch 12, pp 423-432)

2)  Cyclin dependent Kinase 2 (Ch 12, pp 469-472)

3)  Src Protein Kinase (Tyrosine Kinases, p 439-444)

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Learning Objectives

•  Understand Kinase Regulation across family members.

•  Key Features of Active Site and Substrate Binding.

•  Understand the Reaction Mechanism (acid base catalysis) and structure of the Transition State.

•  Know Kinase conserved sequence motifs that define Structural components of the active site.

•  Kinase Autophosphorylation and regulatory consequences.

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Protein Kinase Families

1)  Ser/Thr Kinases

2)  Tyrosine Kinases

3)  Requirement of effector domains and other proteins to activate or repress catalytic activity.

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cAMP Dependent Protein Kinase

Lecture Outline:

•  Subunits and regulation of PKA. •  Substrate Specificity and Consensus Sequence •  Designing Sequence Based Inhibitors •  Three dimensional Structure – The Kinase Fold •  Substrate Binding Cleft

•  Catalytic Mechanism and Sequence Motifs •  Activation/Regulation by Phosphorylation •  Transition State Geometry and Inhibitors

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Cyclic AMP-Dependent Protein Kinase: Signaling and Regulation

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PKA (cAMP-dependent Protein Kinase) Catalytic Domain and Inhibitor

N-domain is Blue (1-126). C-domain is Orange (127-159; and 204-322). Inhibitor Peptide is Red. Active Site loop and Activation Segment are (161-202). Thr197 is Phosphorylated. From PDB coordinates 1APM. What side chains are shown in this picture?

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Protein Kinase A R-subunit Binding

This is rotated ~90 deg ccw from previous views and also shows binding of R-subunit

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Active Form of Protein Kinase A with Inhibitor + ATP and Mn cations.

N-domain is Blue. C-domain is Orange. Inhibitor Peptide is Red. Active Site loop and Activation Segment are . From PDB coordinates 1ATP. What side chains are shown in this picture?

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Closeup View of Activation Segment and Active Site Loop

The Mn2+ cations (green) mimic Mg2+.

The inhibitor peptide has been removed for clarity. Can you describle the roles of the residues shown in this figure? PDB entry: 1ATP

Lys 72 Glu 91

ATP

Lys 168

Asp 166

Arg 165 Thr 201

Phospho- Thr197

Lys 189

Asn 171

Asp 184

Hairpin in middle of activation loop, located between N- and C-domains at back of molecule.

N-Domain

Activation Segment

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Closeup View of Glycine-Rich Hairpin and ATP

The β-hairpin from residues 49-57 acts as a flexible lid that sits over the ATP phosphate groups. GTGSFGRV The backbone N-H groups of residues 53, 54 and 55 make N-H ...O hydrogen bonds with the two terminal phosphates of ATP. PDB entry: 1ATP

Flexible hairpin

Glu127

Lys168

Glu91

Phe54 Val57

Asp184

Gly52

Gly55

Gly50 Lys72

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Mus musculus PKA 12 QESVKEFLAKAKEDFLKKWETPSQNTAQLDQFDRIKTLGTGSFGRVMLVKHKESGNHYAMKILDKQKVVK--- 81 C. elegans PKA 42 AEETHMKLSITPTRESFSLSQLERIITIGKGTFGRVELARDKITGAHYALKVLNIRRVVD--- 101 S. cerevisiae PKA 65 EEQYKQFIAQAR---------VTGGKYSLQDFQILRTLGTGSFGRVHLIRSRHNGRYYAMKVLKKEIVVR--- 125 H. sapiens CDK2 2 ENFQKVEKIGEGTYGVVYKARNKLTGEVVALKKIRLDTETE--- 42 H. sapiens CDK7 10 KRYEKLDFLGEGQFATVYKARDKNTNQIVAIKKIKLGHRSEAKD 53 H. sapiens LCK 258 DEWEVPRETLKLVERLGAGQFGEVWMGY-YNGHTKVAVKSLKQGSMS---- 303 G. gallus cSRC 258 DAWEIPRESLRLEVKLGQGCFGEVWMGT-WNGTTRVAIKTLKPGNMS---- 303 * * * * * * Mus musculus PKA 82 LKQIEHTLNEKRILQAVNFPFLVKLEFSFKDNSNLYMVMEYVAGGEMFSHLRRIGRFSEP 141 C. elegans PKA 102 MRQTQHVHNEKRVLLQLKHPFIVKMYASEKDSNHLYMIMEFVPGGEMFSYLRASRSFSNS 161 S. cerevisiae PKA 126 LKQVEHTNDERLMLSIVTHPFIIRMWGTFQDAQQIFMIMDYIEGGELFSLLRKSQRFPNP 185 H. sapiens CDK2 43 -GVPSTAIREISLLKELNHPNIVKLLDVIHTENKLYLVFEFLHQDLKKFMDASALTGIPL 100 H. sapiens CDK7 54 -GINRTALREIKLLQELSHPNIIGLLDAFGHKSNISLVFDFMETDLEVIIKDNSLVLTP- 111 H. sapiens LCK 304 ---PDAFLAEANLMKQLQHQRLVRLYAVVTQ-EPIYIITEYMENGSLVDFLKTPSGIKLT 359 G. gallus cSRC 304 ---PEAFLQEAQVMKKLRHEKLVQLYAVVSE-EPIYIVTEYMSKGSLLDFLKGEMGKYLR 359 * *(P-2) Mus musculus PKA 142 HARFYAAQIVL--TFEYLHSLDLIYRDLKPENLLIDQQGYIQVTDFGFAKRVKGRTWTLCGT 201 C. elegans PKA 162 MARFYASEIVC--ALEYIHSLGIVYRDLKPENLMLSKEGHIKMADFGFAKELRDRTYTICGT 221 S. cerevisiae PKA 186 VAKFYAAEVCL--ALEYLHSKDIIYRDLKPENILLDKNGHIKITDFGFAKYVPDVTYTLCGT 245 H. sapiens CDK2 101 -PLIKSYLFQLLQGLAFCHSHRVLHRDLKPQNLLINTEGAIKLADFGLARAFGVPVRTYTHE 162 H. sapiens CDK7 112 -SHIKAYMLMTLQGLEYLHQHWILHRDLKPNNLLLDENGVLKLADFGLAKSFGSPNRAYTHQ 172 H. sapiens LCK 360 INKLLDMAAQIAEGMAFIEERNYIHRDLRAANILVSDTLSCKIADFGLARLIEDNEYTAREG 421 G. gallus cSRC 360 LPQLVDMAAQIASGMAYVERMNYVHRDLRAANILVGENLVCKVADFGLGRLIEDNEYTARQG 421 ** * * *** * ** Mus musculus PKA 202 -----PEYLAPEIILSKGYNK-AVDWWALGVLIYEMAA-GYPPFFADQPIQIYEKIVSGKVRFPSHF 261 C. elegans PKA 222 -----PDYLAPESLARTGHNK-GVDWWALGILIYEMMV-GKPPFRGKTTSEIYDAIIEHKLKFPRSF 281 S. cerevisiae PKA 246 -----PDYIAPEVVSTKPYNK-SIDWWSFGILIYEMLA-GYTPFYDSNTMKTYEKILNAELRFPPFF 306 H. sapiens CDK2 163 --VVTLWYRAPEILLGCKYYSTAVDIWSLGCIFAEMVT-RRALFPGDSEIDQLFRIFRTLGTPDEVV 226 H. sapiens CDK7 173 --VVTRWYRAPELLFGARMYGVGVDMWAVGCILAELLL-RVPFLPGDSDLDQLTRIFETLGTPTEEQ 236 H. sapiens LCK 422 -AKFPIKWTAPEAINY-GTFTIKSDVWSFGILLTEIVTHGRIPYPGMTNPEVIQNLERGYRMVRPDN 485 G. gallus cSRC 422 -AKFPIKWTAPEAALY-GRFTIKSDVWSFGILLTELTTKGRVPYPGMVNREVLDQVERGYRMPCPPE 485 * **(P-6) * P+1 pocket P-2 pocket

N-domain

C-domain Part 1

Catalytic Loop Act. Segment

C-domain Part 2

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Mechanism of Activation of PKA

Asp166 functions as a general base. Lys72 binds α and β phosphates of ATP. Glu91 positions Lys 72 (how?). Asp184 binds Mg2+ Mg2+ binds ATP β and γ PO4 oxygen atoms. Arg165 and Lys189 bind Thr197-PO4

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Function Protein Kinase A CDK2 cSRC Binds ATP αβPO4 Lys 72 Lys 33 Lys 295 Salt Bridge to Lys 72 Glu 91 Glu 51 Glu 310 ATP Ribose H-bond Glu 127 Asp 86 None

General Base Asp 166 Asp 127 Asp 386 Orients Asp 166 Asn 171 Asn 132 Asn 391 Orients Asp 166 Thr 201 Thr 165 None Binds Thr /Tyr PO4 Arg 165 Arg 126 Arg 385 Binds ATP γPO4 Lys 168 Lys 129 Arg 388 Phosphorylated Thr 197 Thr 160 Tyr 416 Binds Thr/Tyr PO4 Lys 189 Arg 150 Arg 409 Binds Mg2+ Asp 184 Asp 145 Asp 404

Conserved Residues Important for Protein Kinase Function

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Inhibitor Binding to Protein Kinase A Serine-Based Peptide L R R A S L G KM = 16.0 µM Ala-Based Peptide L R R A A L G Ki = 490 µM PKI(5-24) T T Y A D F I A S G R T G R R N A I H D Ki = 2.3 nM 12 10 9 8 7 6 5 4 3 2 1 0 1 2 3 - + Helix Turn Extended

Note the conservation of Arg residues at positions P-2, P-3 and P-6 on the inhibitor peptide. Arg at P-2 makes a strong salt bridge to Glu170 (plus a weaker one to Glu230) on the Kinase. The side chain of Glu170 also H-bonds the backbone NH of the inhibitor peptide at residue P-2. Arg at P-3 makes a strong salt bridge to Glu127. Glu127 also H-bonds to the ATP ribose and is moderately conserved in Ser/Thr Kinases. Arg at P-6 makes a salt bridge to Glu203, part of the activation loop.

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Substrate Binding Surface in Protein Kinase A

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Mus musculus PKA 12 QESVKEFLAKAKEDFLKKWETPSQNTAQLDQFDRIKTLGTGSFGRVMLVKHKESGNHYAMKILDKQKVVK--- 81 C. elegans PKA 42 AEETHMKLSITPTRESFSLSQLERIITIGKGTFGRVELARDKITGAHYALKVLNIRRVVD--- 101 S. cerevisiae PKA 65 EEQYKQFIAQAR---------VTGGKYSLQDFQILRTLGTGSFGRVHLIRSRHNGRYYAMKVLKKEIVVR--- 125 H. sapiens CDK2 2 ENFQKVEKIGEGTYGVVYKARNKLTGEVVALKKIRLDTETE--- 42 H. sapiens CDK7 10 KRYEKLDFLGEGQFATVYKARDKNTNQIVAIKKIKLGHRSEAKD 53 H. sapiens LCK 258 DEWEVPRETLKLVERLGAGQFGEVWMGY-YNGHTKVAVKSLKQGSMS---- 303 G. gallus cSRC 258 DAWEIPRESLRLEVKLGQGCFGEVWMGT-WNGTTRVAIKTLKPGNMS---- 303 * * * * * * Mus musculus PKA 82 LKQIEHTLNEKRILQAVNFPFLVKLEFSFKDNSNLYMVMEYVAGGEMFSHLRRIGRFSEP 141 C. elegans PKA 102 MRQTQHVHNEKRVLLQLKHPFIVKMYASEKDSNHLYMIMEFVPGGEMFSYLRASRSFSNS 161 S. cerevisiae PKA 126 LKQVEHTNDERLMLSIVTHPFIIRMWGTFQDAQQIFMIMDYIEGGELFSLLRKSQRFPNP 185 H. sapiens CDK2 43 -GVPSTAIREISLLKELNHPNIVKLLDVIHTENKLYLVFEFLHQDLKKFMDASALTGIPL 100 H. sapiens CDK7 54 -GINRTALREIKLLQELSHPNIIGLLDAFGHKSNISLVFDFMETDLEVIIKDNSLVLTP- 111 H. sapiens LCK 304 ---PDAFLAEANLMKQLQHQRLVRLYAVVTQ-EPIYIITEYMENGSLVDFLKTPSGIKLT 359 G. gallus cSRC 304 ---PEAFLQEAQVMKKLRHEKLVQLYAVVSE-EPIYIVTEYMSKGSLLDFLKGEMGKYLR 359 * *(P-2) Mus musculus PKA 142 HARFYAAQIVL--TFEYLHSLDLIYRDLKPENLLIDQQGYIQVTDFGFAKRVKGRTWTLCGT 201 C. elegans PKA 162 MARFYASEIVC--ALEYIHSLGIVYRDLKPENLMLSKEGHIKMADFGFAKELRDRTYTICGT 221 S. cerevisiae PKA 186 VAKFYAAEVCL--ALEYLHSKDIIYRDLKPENILLDKNGHIKITDFGFAKYVPDVTYTLCGT 245 H. sapiens CDK2 101 -PLIKSYLFQLLQGLAFCHSHRVLHRDLKPQNLLINTEGAIKLADFGLARAFGVPVRTYTHE 162 H. sapiens CDK7 112 -SHIKAYMLMTLQGLEYLHQHWILHRDLKPNNLLLDENGVLKLADFGLAKSFGSPNRAYTHQ 172 H. sapiens LCK 360 INKLLDMAAQIAEGMAFIEERNYIHRDLRAANILVSDTLSCKIADFGLARLIEDNEYTAREG 421 G. gallus cSRC 360 LPQLVDMAAQIASGMAYVERMNYVHRDLRAANILVGENLVCKVADFGLGRLIEDNEYTARQG 421 ** * * *** * ** P+1 Pocket Mus musculus PKA 202 -----PEYLAPEIILSKGYNK-AVDWWALGVLIYEMAA-GYPPFFADQPIQIYEKIVSGKVRFPSHF 261 C. elegans PKA 222 -----PDYLAPESLARTGHNK-GVDWWALGILIYEMMV-GKPPFRGKTTSEIYDAIIEHKLKFPRSF 281 S. cerevisiae PKA 246 -----PDYIAPEVVSTKPYNK-SIDWWSFGILIYEMLA-GYTPFYDSNTMKTYEKILNAELRFPPFF 306 H. sapiens CDK2 163 --VVTLWYRAPEILLGCKYYSTAVDIWSLGCIFAEMVT-RRALFPGDSEIDQLFRIFRTLGTPDEVV 226 H. sapiens CDK7 173 --VVTRWYRAPELLFGARMYGVGVDMWAVGCILAELLL-RVPFLPGDSDLDQLTRIFETLGTPTEEQ 236 H. sapiens LCK 422 -AKFPIKWTAPEAINY-GTFTIKSDVWSFGILLTEIVTHGRIPYPGMTNPEVIQNLERGYRMVRPDN 485 G. gallus cSRC 422 -AKFPIKWTAPEAALY-GRFTIKSDVWSFGILLTELTTKGRVPYPGMVNREVLDQVERGYRMPCPPE 485 * **(P-6) * P+1 pocket P-2 pocket

N-domain

C-domain Part 1

Catalytic Loop Act. Segment

C-domain Part 2

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Representation of Transition State in Phosphoryl Transfer

Here, a phosphate would be undergoing inversion of configuration, so phosphorous would be where the carbon is located.

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Protein Kinase A with Bound AlF3 ADP and Substrate Peptide

AlF3 mimics the transition state, exactly ½ way along the “umbrella” flipping pathway of PO4 group inversion. (SN2 reaction)