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Cyclic nucleotides (cNMPs) play a central role as second messengers in signal transduction. Proteins involved in cyclic nucleotide signaling need to be capable of sensing subtle changes in cNMP concentrations. Specialized domains bind cyclic nucleotides such as cAMP or cGMP to transduce signals into physiological responses. The major mammalian effector proteins for cNMP signaling encompass PKA, PKG, the exchange protein directly activated by cAMP (EPAC), hyperpolarization-activated cyclic nucleotide–gated cation channels (HCN) as well as cyclic nucleotide–gated ion channels (CNG). All these effectors share a highly conserved cyclic nucleotide binding (CNB) domain. The CNB domain undergoes major conformational changes upon binding of the cyclic nucleotide and shows two distinct structures for its inactive apo state and its active bound state. These underlying conformational rearrangements alter protein–protein interactions and thus generate a biological response. We focus on unraveling the underlying molecular mechanisms using a combination of mutational approaches, cNMP analogs (Development of cyclic nucleotide analogs for EPAC, HCN and cdiGMP-binding molecules) and structural analyses in collaboration with others.

  • Kim JJ, Flueck C, Franz E, Sanabria-Figueroa E, Thompson E, Lorenz R, Bertinetti D, Baker DA, Herberg FW, Kim C. Crystal Structures of the Carboxyl cGMP Binding Domain of the Plasmodium falciparum cGMP-dependent Protein Kinase Reveal a Novel Capping Triad Crucial for Merozoite Egress.PLoS Pathog. 2015 Feb 3;11(2):e1004639. doi: 10.1371/journal.ppat.1004639. eCollection 2015 Feb.
  • Huang GY, Kim JJ, Reger AS, Lorenz R, Moon EW, Zhao C, Casteel DE, Bertinetti D, Vanschouwen B, Selvaratnam R, Pflugrath JW, Sankaran B, Melacini G, Herberg FW, Kim C.,* Structural Basis for Cyclic-Nucleotide Selectivity and cGMP-Selective Activation of PKG Structure. Structure. 2014 Jan 7;22(1):116-24. doi: 10.1016/j.str.2013.09.021.
  • Möller S, Alfieri A, Bertinetti D, Aquila M, Schwede F, Lolicato M, Rehmann H, Moroni A, Herberg FW. Cyclic nucleotide mapping of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels ACS Chem Biol. 2014 May 16;9(5):1128-37. doi: 10.1021/cb400904s. Epub 2014 Mar 7
  • Herberg FW, Dostmann WR, Zorn M, Davis SJ, Taylor SS. Crosstalk between domains in the regulatory subunit of cAMP-dependent protein kinase: influence of amino terminus on cAMP binding and holoenzyme formation. Biochemistry. 1994 Jun 14;33(23):7485-94
  • Su, Y., Dostmann, W.R.G., Herberg, F.W., Durick, K., Xuong, N-h, Taylor, S.S. and Varughese, K.I. (1995) Regulatory Subunit of  Protein Kinase A: Structure of a Deletion Mutant with cAMP Binding Domains Science 269: 807-813

To understand molecular mechanisms of cyclic nucleotide (cNMP) binding, we use cyclic nucleotides as tools for functional proteomics and in direct and indirect binding studies. We also use tools to directly target cNMP mediated signaling processes in living cells. In collaboration with the company Biolog, we have identified numerous agonists and antagonists with high specificity and isoform selectivity to address PKA, PKG, EPAC and HCN channels.

  • Schwede F, Bertinetti D, Langerijs CN, Hadders MA, Wienk H, Ellenbroek JH, de Koning EJ, Bos JL, Herberg FW, Genieser HG, Janssen RA, Rehmann H. Structure-guided design of selective Epac1 and Epac2 agonists PLoS Biol. 2015 Jan 20;13(1):e1002038. doi: 10.1371/journal.pbio.1002038. eCollection 2015 Jan
  • Isensee J, Diskar M, Waldherr S, Buschow R, Hasenauer J, Prinz A, Allgöwer F, Herberg FW, Hucho T Pain modulators regulate the dynamics of PKA-RII phosphorylation in subgroups of sensory neurons J Cell Sci. 2014 Jan 1;127(Pt 1):216-29. doi: 10.1242/jcs.136580
  • Frank Schwede, Oleg G. Chepurny, Melanie Kaufholz, Daniela Bertinetti, Colin A. Leech, Over Cabrera, Yingmin Zhu, Fang Mei, Xiaodong Cheng, Hans-G. Genieser, F.W. Herberg, and George G. Holz “Rp-cAMPS prodrugs reveal the cAMP dependence of rat first-phase glucose-stimulated insulin secretion” accepted with revisions Mol. Endokrin
  • Diskar M, Zenn, H.M., Kaupisch, A., Prinz, A., Herberg, F.W. Molecular basis for isoform specific autoregulation of protein kinase A - insights from the human catalytic subunit PrKX (2007) Cell Signal. 2007 Oct;19(10):2024-34

Coordinated by: Prof. S. S. Taylor and Prof. F. W. Herberg
Funding period: since 2016
Funding: Michael J Fox Foundation for Parkinson’s Researc

Parkinson’s disease (PD) is the most common neurodegenerative movement disorder. In the last decade several gene loci have been linked to the pathogenesis of PD, among them the PARK8 locus that encodes for the Leucine-Rich Repeat Kinase 2 (LRRK2). LRRK2 has been associated with both, sporadic as well as inherited PD suggesting a central role in disease pathology. LRRK2 exhibits both, kinase as well as GTPase activity and also acts as a scaffold (figure 1). Several mutations in LRRK2 have been correlated to familial PD, but despite significant research efforts, little is known about the regulation of this 285 kDa multi-domain phosphoprotein.

Funded by the Michael J Fox Foundation for Parkinson’s Research and the Otto Braun Fund we study the molecular basis of LRRK2 regulation, addressing both, the enzymatic and the scaffolding functions. This allows us to achieve a better mechanistic understanding of the functional consequences of PD-relevant mutations. Applying innovative concepts like kinase spines and employing novel tools, i.e. nanobodies, we want to investigate the interplay of the GTPase domain with the protein kinase activity.

In addition, we study the binding of known interaction partners such as 14-3-3-isoforms and their influence on conformation and activity of LRRK2. We already described an essential function for cAMP-dependent protein kinase (PKA) and 14-3-3 interaction in the negative regulation of LRRK2 kinase activity (Muda et al. PNAS 2014).
Employing a combination of structural, molecular, biochemical, cellular and biophysical techniques will help us to describe the modulation of LRRK2 function in healthy and diseased states. Understanding LRRK2 regulation on a molecular level will not only provide insights into mechanisms of PD neurodegeneration but may also facilitate the development of therapeutics.

Figure 1: LRRK2 protein domain organization. LRRK2 is composed of an N-terminal Armadillo (Arm) domain, followed by Ankyrin repeats (Ank) and the name giving leucine-rich repeats (LRR), ROC (Ras of complex, a GTPase), COR (C-terminal of ROC), a kinase domain and WD40 repeats at the C-terminus. Most common pathogenic amino acid substitutions including R1441C/G/H/S, Y1699C I2020T and G2019S are highlighted. The sequence and position of a 14 3 3 interaction motif in LRRK2 is shown.

Coordinated by: Prof. F. W. Herberg, Kassel, Germany
Funding period: since 2016
Funding number: DFG, Projektnummer 322864367

The parasite Plasmodium falciparum is estimated to be responsible for 99% of the malaria infections in sub-Saharan Africa. Although medications are available, an ongoing resistance development is observed. The activity of the cGMP-dependent protein kinase of P. falciparum (PfPKG) is a key player in controlling the life cycle of this parasite. Downregulation or inhibition of PfPKG has shown to successfully kill the organism alongside with blocking red blood cell egress and evasion. Targeting PfPKG therefore represents a promising possibility for alternative malaria treatments.
We recently have demonstrated that the C-terminal cyclic nucleotide binding domain (CNB-D) of PfPKG is crucial for activation (Franz et al 2018). We are screening a set of differently modified cGMP analogues regarding their binding kinetics, affinity and activation as well as their usability as potential antagonists for PfPKG.
Major differences in the three-dimensional structure of PfPKG compared to humanPKG are based on primary sequence alignments (Figure1 ) and crystal structures. Furthermore, PfPKG seems to undergo unusual large conformational changes upon cGMP-binding (Figure 2) as indicated by analytical gel filtration.

Figure 1: (a) Domain organization of the cGMP-dependent protein kinase of Plasmodium falciparum (PfPKG) and (b) of human cGMP-dependent protein kinases (hPKG). CNB: Cyclic nucleotide binding domain; ATP: small lobe of the catalytic domain; LZ: leucine zipper; AS: autoinhibitory sequence; substrate: large lobe of the catalytic domain.
Figure 2: Putative activation mechanism based on structural data for the cGMP-free PfPKG (pdb: 5DYK). Gel filtration experiments indicating large structural rearrangements in upon cGMP binding to the PfPKG regulatory domain.

Coordinated by: Mr. Alper Karakaya, Düzen Biological Sciences R&D and Production, Turkey
Funding period: since 2018
Funding: ERA CoBioTech

The ERA CoBioTech project RHODOLIVE (biovalorization of olive mill waste water to microbial lipids and other products via Rhodotorula glutinis fermentation) is funded by an ERA-Net Cofund Action aiming to strengthen the European research area in biotechnology. The main objective of the project is to develop a sustainable bioprocess for olive mill waste water bioremediation with R. glutinis to produce high value-added bioproducts including carotenoids, bioactive phenolic compounds, lipids etc. The demonstration in our project will focus on food products, meanwhile these compounds are also frequently used in cosmetics and pharmaceuticals.

The role of physiologically relevant metal ions on protein kinase inhibition and activity has been underestimated so far. PKA among other kinases strongly prefers magnesium as a co-factor. Other physiologically important divalent metal ions like calcium or zinc cannot support steady-state catalysis. Measurements, performed using classical Michaelis-Menten conditions, led to the notion that calcium is not able to assist in phosphoryl-transfer. Recent crystal structures, however, showed complete phosphoryl-transfer, even in the presence of calcium. We could show that indeed is able to assist in phosphoryl transfer, yet hampers the release of the products. This has bee described for other (patho)physiological metal ions (manganese, zinc and cadmium). Abundance of different metal ions thus may act as an additional regulatory mechanism for kinase activity. In particular PKA is localized directly in the vicinity of calcium channels, our results strongly suggest that PKA activity is susceptible to fluctuations in calcium concentrations. This provides a novel link between cAMP- and calcium signaling.

  • Herberg, F.W. and Taylor, S.S. (1993) Physiological Inhibitors of the Catalytic Subunit of cAMP-dependent Protein Kinase: Effect of MgATP on Protein/Protein Interaction.  Biochemistry  32:14015 -14022
  • Zimmermann B, Schweinsberg S, Drewianka S, Herberg FW. Effect of metal ions on high-affinity binding of pseudosubstrate inhibitors to PKA. Biochem J 2008, 413(1):93-101
  • Zhang, P., Matthias J. Knape, Lalima G. Ahuja, Malik M. Keshwani, Charles C. King, Mira Sastri, F.W. Herberg, S.S. Taylor “Single Turnover Signaling Cycle of the PKA RIIβ Holoenzyme” PLoS Biol 13(7): e1002192. doi:10.1371/journal.pbio.1002192 d
  • Knape, MJ, Ahuja, LG, Bertinetti, D, Burghardt, NCG, Zimmermann, B, Taylor, SS, Herberg, FW Divalent metal ions Mg2+ and Ca2+ have distinct effects on protein kinase A activity and regulation ACS Chem Biol. 2015 Oct 16;10(10):2303-15. doi: 10.1021/acschembio.5b00271
  • Knape MJ, Ballez M, Burghardt NC, Zimmermann B, Bertinetti D, Kornev AP and Herberg FW Divalent metal ions control activity and inhibition of protein kinases Metallomics 2017 2017 Nov 15;9(11):1576-1584
  • Knape MJ and Herberg FW Metal coordination in kinases and pseudokinases Biochemical Society Transactions (2017) Jun 15;45(3):653-663. doi: 10.1042/BST20160327

The assembly of multi-protein complexes orchestrates protein kinase activity in a spatial and temporal manner. One family of scaffolding proteins involeved are A-kinase anchoring proteins (AKAPs). AKAPs share the commonality of binding PKA but are highly divergent in terms of function. In addition, they bind other signaling proteins and kinase substrates and tether such multi-protein complexes to subcellular locations. We have established unique techniques to quantitatively characterize isoform-specific AKAP interactions using SPR and FP. In addition, we have collaborated closely with E. Kennedy (UGA, Athens GA) and E. Klussmann (MDC Berlin) to develop and characterize strategies for targeted disruption of AKAP complexes using stapled peptides.

  • Bendzunas NG, Dörfler S, Autenrieth K, Bertinetti D, Machal EMF, Kennedy EJ, Herberg FW. Investigating PKA-RII specificity using analogs of the PKA:AKAP peptide inhibitor STAD-2. Bioorg Med Chem. 2018 Feb 12. pii: S0968-0896(17)31514-6. doi: 10.1016/j.bmc.2018.02.001
  • Hermann JS, Skroblin P, Bertinetti D, Hanold LE, von der Heide EK, Wagener EM, Zenn HM, Klussmann E, Kennedy EJ, Herberg FW. “Neurochondrin is an atypical RIIα-specific A-kinase anchoring protein” Biochim Biophys Acta. 2015 Apr 23. pii: S1570-9639(15)00115-6. doi: 10.1016/j.bbapap.2015.04.018
  • Götz F, Roske Y, Schulz MS, Autenrieth K, Bertinetti D, Faelber K, Zühlke K, Kreuchwig A, Kennedy E, Krause G, Daumke O, Herberg FW, Heinemann U, Klussmann E. AKAP18: PKA-RIIα structure reveals crucial anchor points for recognition of regulatory subunits of PKA Biochem J. 2016 Apr 21. pii: BCJ20160242
  • Autenrieth K, Bendzunas G, Bertinetti D, *Herberg FW, *Kennedy EJ.  Defining A-Kinase Anchoring Protein (AKAP) Specificity for Protein Kinase A Subunit RI (PKA-RI). Chembiochem. 2016 Apr 15;17(8):693-7. doi: 10.1002/cbic.201500632
  • Wang Y, Ho TG, Bertinetti D, Neddermann M, Franz E, Mo GC, Schendowich LP, Sukhu A, Spelts RC, Zhang J, Herberg FW, Kennedy EJ.Isoform-selective disruption of AKAP-localized PKA using hydrocarbon stapled peptides. ACS Chem Biol. 2014 Mar 21;9(3):635-42
  • Wang Y, Ho TG, Franz E, Hermann JS, Smith FD, Hehnly H, Esseltine JL, Hanold LE, Murph MM, Bertinetti D, Scott JD, Herberg FW, Kennedy EJ. “PKA-type I Selective Constrained Peptide Disruptors of AKAP Complexes” ACS Chem Biol. 2015 Mar 25