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Protein Structure and Molecular Modeling Laboratory

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Research Staff:

Metaxia Vlassi, PhD, Researcher A'

 Christoforos Zarkadas, Msc, Computer Scientist

Research Interests

  • Protein folding

  • Conformation of aa-sequence repeats and repeat-mediated protein interactions

  • Conformational changes and protein stability

  • Phosphorylation and conformational changes

  • Molecular dynamics simulations of proteins

  • Protein-Protein  interactions

  • Intrinsically disordered proteins
  •  Structure & dynamics of enzymes as potential therapeutic targets 


Our current research activities focus on

  • Conformational studies of proteins with emphasis on sequence-repeat (/protein interaction modules) containing proteins aiming at elucidating their sequence/structure relationships and at unraveling the atomic details of repeat-mediated interactions towards a full understanding of protein-protein interaction mechanisms

  • Studying the dynamics of protein structure by means of molecular dynamics (MD) simulations and development of related bio-computing tools.

  • In silico structural studies of enzymes as potential therapeutic targets and of their interactions aiming at unraveling the atomic details of their activation/inhibition mechanisms towards a full understanding of their function as a tool for rational development on new therapeutics.

The approach we follow includes, mainly, in silico approaches (such as homology/comparative modeling and threading, molecular dynamics simulations, molecular docking etc) combined with biochemical and biophysical (mainly circular dichroism (CD) spectroscopy) data.


  • Conformational Preferences of amino-acid sequence Repeats, Role in Protein Interactions


Sequence repetition is very common in proteins involved in multiple protein interactions, with a wide variety of functions. Despite their abundance and importance however, questions about the conformational preferences of many classes of repeats and the structural determinants of repeat-mediated interactions, remain open. For example, arginine/serine (RS) repeats are found in several proteins in metazoans with a wide variety of functions, many of which are regulated by SR protein kinase 1 (SRPK1)-mediated phosphorylation. However, their conformational preferences as well as their recognition mechanism by SRPK1 remain rather elusive. Another example of structurally uncharacterized repeats is that of short tandem repeats, recently identified (by our collaborator, M. Andrade-Navaro and co-workers, Berlin, Germany) in the inhibitory domain of the human mineralocorticoid receptor (MR) and of selected orthologs. Finally, even in the case of well characterized repeats, such as the tetratricopeptide repeats (TPR), questions about their structural stability and their interaction mechanism, remain open.

1. Short tandem repeats in the inhibitory domain of the mineralocorticoid receptor: prediction of a beta-solenoid structure

The human mineralocorticoid receptor (MR) is one of the main components of the renin-angiotensin-aldosterone system (RAAS), the system that regulates the body exchange of water and sodium. The MR has two trans-activating ligand-independent domains and one inhibitory domain (ID), which modulates the activity of the former. Although it is known that prior folding of the MR-ID is required for binding of several transcriptional co-regulatory proteins, its detailed 3D-structure was unknown, so far.

Using in silico approaches, we very recently, modelled the 3D-structure of five consecutive repeats from the ensemble of short tandem repeats identified in the human MR-ID. Based on our model (Figure 1), we propose that the repeat region of the MR-ID is compatible with a β-helical fold offering a scaffold for multiple intra- and inter-molecular interactions (including dimerization) of the MR receptor and that these interactions are modulated by phosphorylation-dependent conformational changes, regulated by specific kinases, thus playing an important role in the coordination and sequential interactions of various MR partners and therefore in the specificity and the (patho)physiological function of this receptor. (For details see: Vlassi et al, 2013 [PubMed])

2. Arginine-Serine (RS) Repeats:

Using in silico molecular modeling techniques (3D-modeling and MD simulations) on the short RS-domain of lamin-B receptor (LBR), combined with biochemical and literature data, we showed recently that, unphosphorylated RS repeats adopt short, transient helical conformations, whereas Ser-phosphorylation induces more compact (Arg-claw-like) structures (Figure 2), irrespective of the repeat length, probably serving in recognition of basic partners, such as is histone H3 for LBR. In addition, we showed, for the first time, that even short RS-domains may be constituents of recognition platforms for SRPK1, which in turn, uses the same, distal to the active site, acidic docking groove (Figure 3) to recognize its RS substrates, irrespective of their length. In total, our results shed light on the conformational preferences of an important class of sequence repeats before and after their phosphorylation, as well as on aspects of their recognition by SRPK1 and support the idea that the RS repeats share a common recognition mechanism by SRPK1, irrespective of their length, thus adding to knowledge towards a full understanding of their phosphorylation and interaction mechanisms. (For details see: Sellis et al. 2012 [PubMed])

3. Tetratricopeptide Repeats (TPR)

TPR motifs have been found in a wide variety of proteins in all organisms and are known to mediate multiple protein-protein interactions. Ssn6, a tetratricopeptide repeat (TPR) containing protein, associates with the Tup1 repressor to form a global transcriptional co-repressor complex, which is conserved across species. In the past (Gounalaki et al 2000 [PubMed)), using the first crystallographic structure of a TPR protein as template, we have produced a 3D-model of the TPR part of the Tup1-binding domain of Ssn6, which combined with mutagenesis data (in collaboration with T. Tzamarias, IMBB, Crete) revealed that, in its bound form, the Tup1-binding domain of Ssn6 follows the typical TPR, α-helical coiled-coil, configuration. Our subsequent research efforts, including a combination of circular dichroism (CD) spectroscopy, limited proteolysis mapping and in silico techniques, revealed that in the absence of Tup1, the Tup1-binding domain of Ssn6 is partially unfolded, whereas a conformational change, involving a disorder-to-helix transition and stabilization of the typical TPR coiled-coil configuration, occurs upon Tup1 binding. In total these two studies, reinforce the notion of an induced-fit mechanism for this particular TPR-interaction and suggest that folding-coupled-to-binding may be more common in TPR-mediated interactions than previously believed (For details see: Palaiomyltiou et al 2008 [PubMed]))

  • 3D-Modelling of Enzymes as potential therapeutic targets. PARN/inhibitors interactions

Poly(A)-specific ribonuclease (PARN) is a 30-exoribonuclease that efficiently degrades poly(A) tails and regulates, in part, mRNA turnover rates. Human PARN represents a potential target for drug development that had not been extensively studied so far. By means of in silico molecular docking followed by MD simulations, we have previously shown (Balatsos et al 2009, [PubMed]) that synthetic nucleoside analogues with adequate tumour-inhibitory effect, also shown to inhibit PARN in vitro, can dock into the active site of this enzyme. Our more recent in silico docking data on new synthetic nucleoside analogues, shown to be competitive PARN inhibitors, revealed the atomic details of their inhibition mechanisms and clarified the structural determinants of the inhibition mechanism of the most effective compound (uracil-based analogue, named U1), shown to act through slow-release from the active site of the enzyme (Figure 4). In combination, these two docking studies reinforce the notion that PARN can be established as a novel molecular target of structure-based drug design and identify compound U1 as lead compound for further rational development of new more potent PARN inhibitors as potential anticancer agents (For details see: Balatsos et al. 2012, [PubMed]),



Previously, and in the framework of an internal program of NCSR “D” (Demoerevna), we have developed the GROMITA software (Sellis. et al 2009, [PubMed]), which is a graphical user interface (GUI) to GROMACS-4: a suite of programs for performing and analyzing molecular simulations. The original GROMITA version, v.1, is compatible with GROMACS 4.0.x versions. More recently, we developed an updated version, (v.3.01), of the program which is compatible with GROMACS-4 versions 4.5.x. GROMITA (both versions) can be downloaded (license agreement required) from



Zaganas IV, Kanavouras K, Borompokas N, Arianoglou G, Dimovasili C, Latsoudis H, Vlassi M, Mastorodemos V (2014) The odyssey of a young gene: structure-function studies in human glutamate dehydrogenases reveal evolutionary-acquired complex allosteric regulation mechanisms. Neurochem Res. 2014 Mar;39(3):471-86. doi: 10.1007/s11064-014-1251-0. Epub 2014 Feb 11 [PubMed]

Vlassi M*, Brauns, K, Andrade-Navarro MA (2013) Short tandem repeats in the inhibitory domain of the mineralocoricoid receptor: prediction of a beta-solenoid structure. BMC Struct Biol 13:17 [PubMed]

Sellis D, Drosou V, Vlachakis D, Voukkalis N, Giannakouros T, Vlassi M. (2012) Phosphorylation of the arginine/serine repeats of lamin B receptor by SRPK1-Insights from molecular dynamics simulations. Biochim Biophys Acta (General Subjects) 1820(1):44-55. [PubMed]

Balatsos N, Vlachakis D, Chatzigeorgiou V, Manta S, Komiotis D, Vlassi M, Stathopoulos C (2012) Kinetic and in silico analysis of the slow-binding inhibition of human poly(A)-specific ribonuclease (PARN) by novel nucleoside analogues. Biochimie. 94(1):214-21 [PubMed]

Balatsos, N., Vlachakis, D., Maragozidis, P., Anastasakis, D., Kyritsis, A., Vlassi, M., Koumiotis, D., Stathopoulos, C (2009) Competitive Inhibition of Human Poly(A)-Specific Ribonuclease (PARN) by Synthetic Fluoro-Pyranosyl Nucleosides. Biochemistry 48(26):6044-6051 [PubMed]

Sellis, D., Vlachakis, D., Vlassi, M. (2009) Gromita: A fully integrated graphical user interface to Gromacs 4. Bioinformatics & Biology Insights 2009:3, 99-102 [PubMed].

Palaiomylitou, M., Tartas, A, Vlachakis, D., Tzamarias, D., Vlassi, M. (2008) Investigating the structural stability of the Tup1 interaction domain of Ssn6: Evidence for a conformational change in the complex. Proteins 1;70 (1):72-82 [PubMed]