Various Strategies Used to Obtain Proteins for Crystallization and Biostructural Studies Dorothee Ambrosius, R. Engh, F. Hesse, M. Lanzendörfer, S. Palme, P. Rüger Roche Pharmaceutical Research, Penzberg

Protein Classes extracellular proteins  plasma protein concentration:  70 mg/ml transporter (albumin) immuno-globulin enzymes, enzyme-inhibitors coagulation factors, lipoproteins  protein characteristics/ stability often monomeric proteins contain disulfide bridges protease resistant stable fold intracellular proteins  cytoplasma and organelles:  300-800 mg/ml multi-enzyme complexes enzyme cascades transcription complexes focal adhesion/integrins cytoskeleton, heat-shock proteins  protein characteristics/stability often multimeric complexes no disulfide bridges very labile proteins; short half-life require stabilization: interaction with other proteins

Protein Sources/Expression Systems

Biological Function of Cytokines G-CSF Neutrophils Source: Herrmann/Lederle

Development Goals for Recombinant Human G-CSF  Hu-G-CSF: hematopoietic growth factor (174 aa) 2 S-S bridges, one single Cys 17  Clinical use: patients with neutropenia: after chemotherapy  improved haemotopoietic recovery  reduction of infectious risks  native sequence:  without additional N-terminal Met  reduction of immunogenicity risk  potency:  equal to Amgen´s Neupogen  low production cost:  E. coli as host strain  in vitro refolding  consistent quality:  robust downstream scheme  analytical methods established

 Strategy: Development of Recombinant Human G-CSF Fusion Peptide Human G-CSF Fusion Peptide high level expression improved refolding efficient separation of cleaved and uncleaved protein optimized cleavage site Protease specific efficient recombinant consistent quality rhG-CSF low production costs without N-terminal Met equal potency/efficiency consistent quality improved quality Genetic engineering of an economic downstream process

Optimization of rhG-CSF Fusion Proteins Fusion Peptide Met G-CSF Met-Thr-Pro-Leu  G-CSF Met-Thr-Pro-Leu-His-His  G-CSF Met-Thr-Pro-Leu-Lys-Lys  G-CSF Met-Thr-Pro-Leu-Glu-Glu-Gly  G-CSF Met-Thr-Pro-Leu-Glu-Glu-Gly-Thr-Pro-Leu  G-CSF Met-Lys-Ala-Lys-Arg-Phe-Lys-Lys-His  G-CSF Expression (%) 100 30 25 10 Cleavage - ++ + +++ Renaturation (%) 10 20 50 90 80 Cleavage Site (Pro-Arg-Pro-Pro) Source: EP 92102864.3 ; DE 4104580

Refolding Kinetics of rhG-CSF Fusion Protein Solubilization 6,0 M Gdn/HCl, pH 8.0 100 mM Tris,/HCl 100 mM DTE 1 mM EDTA Temperature: RT c= 20 mg/ml Renaturation 0,8 M Arginine/HCl 100 mM Tris/HCl, pH 8.0 0.5 / 0.5 mM = GSH / GSSG 10 mM EDTA Protein conc. 0.5 -1.0 mg /ml Time: 1- 2 hours native Pellet SN denat. Source: EP 92102864.3 ; DE 4104580

Role of p53 in cell cycle control:“guardian of the genome” cell cycle arrest: repair defective genes latent p53 active p53 cell type level of p53 extent of DNA damage genetic background activation accumulation stress factors or oncogenic proteins mdm2 apoptosis: kill harmful deregulated cells negative feedback loop !!

Engineering of MDM2 for biostructural purposes The MDM2 oncoprotein is a cellular inhibitor of the p53 tumor suppressor. Goal: Improvement of biophysical properties of HDM2 (human MDM2) by “crystal engineering” Known:  XDM2 (Xenopus laevis MDM2): - better solubility, suitable for biostructural investigations - wrong species and reduced binding affinity  HDM2 (25-108): - high binding affinity to p53 peptide - prone to aggregation, not suitable for biostructural studies Strategy:  use XDM2 as scaffold and humanize its p53-binding site  introduce point mutations in HDM2 to increase solubility  remove flexible ends at both sides of structured p53-binding region

Structure of MDM2/p53-peptide complex Figures taken from Kussie et al., Science 274 (1996) 948. Resolution X-ray structures: human MDM2/p53: 2.6 Å Xenopus MDM2/p53: 2.3 Å p53 mdm2 17-29 26-108

MDM2 variants created by protein engineering 1 26 108 125 185 240 300 330 350 440 491 human MDM2 p53 binding HDM2 (17-125) X-ray published HDM2 (25-108) X-ray HDM2 (25-108) mutants X-ray XDM2 (13-119) X-ray published, NMR XDM2 (13-119) LHI NMR, X-ray XDM2 (21-105) LHI X-ray I50L P92H L95I

Human MDM2: Yields & Upscale Step 15N-labeled non-labeled (LB) (minimal medium) Fermentation 10 L 10 L E. coli (wet weight) 90 g 600 g Inclusion bodies (w.w.) 3.5 g 85 g IB total protein content 1.3 g 30 g MDM2 (50-70% yield) 0.8 g 18 g Renaturation (~25%) 0.2 g 4.5 g MDM2 (Purification) 0.16 g 3.6 g Final product 0.1 g 2.2 g

Crystals of hXDM/peptide Some crystals comply with corporate identity rules Patience might be rewarded hXDM2/phage-peptide hXDM2/p53 peptide Conditions: 0.1 M MES pH 6.2, 4.0 M NaOOCH 3 days after micro seeding at 13 °C 4 months at 4 °C

Protein Kinase Families (incomplete list) I: Ser/Thr-Kinase Families Subfamilies/Structures Ia: Non Receptor Ser/Thr-Kinase familiy cAPK: cAMP dependent protein kinase PKA, PKB, PKC cdks: Cyclin dependent kinase cdk2, cdk4, cdk6 MAPK: Mitogen activated protein kinase Erk, Erk2, Jnk, p38(,,) MLCK: Myosine light chain kinase Twitchin, Titin CK: Casein kinase Ck-1, Ck-2 PhK: Phosphorylase kinase (tetramer: , , , ) PhK CaMK: Calcium/calmodulin dependen kinase CaMK Ib: Receptor Ser/Thr-Kinase family TGF1-R Kinase TGF1-ßR II: Tyr-Kinase Families Subfamilies/Structures IIa: Non receptor Tyr-Kinase family SRC-family SRC, c-SRC, CSK, HCK LCK: humam lymphocyte kinase: LCK, c-Abl IIb: Receptor Tyr-Kinase family EGFR-family: EGFR, ErbB2-4 InsR-family IRK, IGF1R, IRR PDGFR-, CSFR-, Met-, Ron-familiy, FGF1-R, VEGFR-K EphA1….EphB1, Trk A, B, C, etc.

PKA: 2 Å X-ray Structure Further details for crystallization see poster of Ch. Breitenlechner Figure 6: The X-ray structure of Protein Kinase A, shows the structural elements prototypical for the protein kinase family. An N-terminal beta strand (blue arrows) rich domain and a C-terminal alpha-helix (yellow cylinders) rich domain form the overall topology. The relative domain orientations are stablized by three interconnecting protein segments, ATP (here substituted by inhibitor AMP-PNP), and subtrate (here substituted by inhibitory peptide PKI). An activating phosphorylation site on the flexible activation loop causes structural rearrangements at the enzymatic activation site. The features generally present among protein kinases include flexible loop rearrangements controlled by phosphorylation, domain motions controlled by substrates or co-factors, and ATP binding at the domain interface. These features lead to heterogeneities of structure and phosphorylation, which generally hinder protein preparation for crystallization. On the other hand, they also contribute to the uniqueness of individual protein kinases and thus to the likelihood of finding drugs with the desired properties.

PKA: cyclic AMP Dependent Protein Kinase Expression:  E. coli, solubly expressed in phosphorylated, active form  20-50 mg purified protein (10 l fermentation) Purification:  affinity chromatography with inhibitory peptide (PKI) mimicking substrate binding  Ref.: R. Engh & D. Bossemeyer, Adv. Enz. Reg. 41, 2001 Binding Affinity:  20 nM of inhibitory peptide (PKI) Protein:  MW: 35 kDa  Ser/The kinase  monomeric 2 domain (C- and N-lobe) protein without additional regulatory domains (SH2, SH3, etc.)  extended structured C- and N-Terminus, which possibly stabilizes the overall kinase structure Ideal model: Ser/Thr protein kinase inhibitor studies generation of other Ser/The kinase (e.g. PKB, Aurora) structures

Major Components of the Cell Cycle Machinery G0 mitogen induced progression through the cell cycle requires timely controlled activation of different cyclin-dependent kinases (CDKs) cyclins (D, E, A, B), periodically expressed throughout the cycle, are the regulatory subunits of CDKs (activation) members of the p16(INK4)- and p21(KIP)-protein family inhibit CDKs and CDK-cyclin complexes and arrest inappropriate cell cycle progression CDC2 INK4 M cyclin B Mitosis CDK4/6 Cell Cycle cyclin D G2 G1 CDC2 Kip/ Cip DNA Replication cyc. A/B CDK2 S cyclin E CDK2 cyclin A Kip/ Cip

Cyclin Dependent Kinases: CDK2 and CDK4/6 N. Pavletich, JMB 287, 821-828, 1999

Structural investigations of cdks (incomplete list)

Summary  Proteins show a tremendous diversity with respect to - biological function and cellular location - structure, conformation and stability  E. coli is a very attractive expression system with respect to time, yield, costs and production of isotope labeled proteins  Application of in vitro protein refolding is a powerful tool to generate native structured proteins and should be considered as alternative  The protein kinase family is regulated by multiple mechanism and show conformational diversity of catalytic cores; high degree of flexibility - e.g. IRK(3P) and LCK (Tyr kinases) show structural homology to cAPK and cdks (Ser/Thr kinases)  Until today, most kinases successfully applied for structural research are expressed as active P--enzyme in baculo/insect cells; exception PKA

Acknowledgement PEX: S. Kanzler, H. Brandstetter (MPI) MDM2: G. Saalfrank, Ch. Breitenlechner (MPI), U. Jacob (MPI) IL-16: B. Essig , P. Mühlhahn (MPI), T. Holak (MPI) MIA: G. Saalfrank, C. Hergersberg, R. Stoll (MPI), T. Holak (MPI) cAPK: G. Achhammer, E. Liebig, Ch. Breitenlechner (MPI) cdks: H. Hertenberger, J. Kluge, U. Jucknischke G-CSF: S. Stammler, M. Leidenberger, U. Michaelis, T. Zink (MPI), T. Holak (MPI)