Biomedical subjects

Hendrik R A Jonker

Publications and source records attributed to Hendrik R A Jonker.

5 recordsLinked to original sources

PubMed2006

L11 domain rearrangement upon binding to RNA and thiostrepton studied by NMR spectroscopy.

Ribosomal proteins are assumed to stabilize specific RNA structures and promote compact folding of the large rRNA. The conformational dynamics of the protein between the bound and unbound state play an important role in the binding process. We have studied those dynamical changes in detail for the highly conserved complex between the ribosomal protein L11 and the GTPase region of 23S rRNA. The RNA domain is compactly folded into a well defined tertiary structure, which is further stabilized by the association with the C-terminal domain of the L11 protein (L11(ctd)). In addition, the N-terminal domain of L11 (L11(ntd)) is implicated in the binding of the natural thiazole antibiotic thiostrepton, which disrupts the elongation factor function. We have studied the conformation of the ribosomal protein and its dynamics by NMR in the unbound state, the RNA bound state and in the ternary complex with the RNA and thiostrepton. Our data reveal a rearrangement of the L11(ntd), placing it closer to the RNA after binding of thiostrepton, which may prevent binding of elongation factors. We propose a model for the ternary L11-RNA-thiostrepton complex that is additionally based on interaction data and conformational information of the L11 protein. The model is consistent with earlier findings and provides an explanation for the role of L11(ntd) in elongation factor binding.

Anti-Bacterial Agents↗

PubMed2006

The intrinsically unstructured domain of PC4 modulates the activity of the structured core through inter- and intramolecular interactions.

Proteins frequently contain unstructured regions apart from a functionally important and well-conserved structured domain. Functional and structural aspects for these regions are frequently less clear. The general human positive cofactor 4 (PC4), has such a domain organization and can interact with various DNA substrates, transcriptional activators, and basal transcription factors. While essential for the cofactor function, structural and functional knowledge about these interactions is limited. Using biochemical, nuclear magnetic resonance (NMR), and docking experiments, we show that the carboxy-terminal structured core domain (PC4ctd) is required and sufficient for binding to single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and the herpes simplex virion protein 16 (VP16) activation domain (VP16ad). We determined the interaction surfaces within PC4 and showed that VP16 and DNA binding are mutually exclusive. Although the amino-terminal domain of PC4 (PC4ntd) alone is devoid of any bioactivity, it increases the interaction with VP16ad. While it decreases the ssDNA-binding and DNA-unwinding activity, it does not influence dsDNA binding. Structural characterization of this domain showed that it is highly flexible and mostly unstructured both in the free form and in the complex. NMR titration experiments using various protein and DNA substrates of the individual domains and the full-length PC4 revealed local conformational or environmental changes in both the structured and unstructured subdomains, which are interpreted to be caused by inter- and intramolecular interactions. We propose that the unstructured PC4ntd regulates the PC4 cofactor function by specific interactions with the activator and through modulation and/or shielding of the interaction surface in the structured core of PC4ctd.

Binding Sites↗

PubMed2006

Gradual phosphorylation regulates PC4 coactivator function.

The unstructured N-terminal domain of the transcriptional cofactor PC4 contains multiple phosphorylation sites that regulate activity. The phosphorylation status differentially influences the various biochemical functions performed by the structured core of PC4. Binding to ssDNA is slightly enhanced by phosphorylation of one serine residue, which is not augmented by further phosphorylation. The presence of at least two phosphoserines decreases DNA-unwinding activity and abrogates binding to the transcriptional activator VP16. Phosphorylation gradually decreases the binding affinity for dsDNA. These phosphorylation-dependent changes in PC4 activities correlate with the sequential functions PC4 fulfils throughout the transcription cycle. MS and NMR revealed that up to eight serines are progressively phosphorylated towards the N-terminus, resulting in gradual environmental changes in the C-terminal direction of the following lysine-rich region. Also within the structured core, primarily around the interaction surfaces, environmental changes are observed. We propose a model for co-ordinated changes in PC4 cofactor functions, mediated by phosphorylation status-dependent gradual masking of the lysine-rich region causing shielding or exposure of interaction surfaces.

Amino Acid Sequence↗

PubMed2005

Structural properties of the promiscuous VP16 activation domain.

Herpes simplex virion protein 16 (VP16) contains two strong activation regions that can independently and cooperatively activate transcription in vivo. We have identified the regions and residues involved in the interaction with the human transcriptional coactivator positive cofactor 4 (PC4) and the general transcription factor TFIIB. NMR and biochemical experiments revealed that both VP16 activation regions are required for the interaction and undergo a conformational transition from random coil to alpha-helix upon binding to its target PC4. The interaction is strongly electrostatically driven and the binding to PC4 is enhanced by the presence of its amino-terminal domain. We propose models for binding of VP16 to the core domains of PC4 and TFIIB that are based on two independent docking approaches using NMR chemical shift changes observed in titration experiments. The models are consistent with results from site-directed mutagenesis and provide an explanation for the contribution of both acidic and hydrophobic residues for transcriptional activation by VP16. Both intrinsically unstructured activation domains are attracted to their interaction partner by electrostatic interactions, and adopt an alpha-helical conformation around the important hydrophobic residues. The models showed multiple distinct binding surfaces upon interaction with various partners, providing an explanation for the promiscuous properties, cooperativity, and the high activity of this activation domain.

Herpes Simplex Virus Protein Vmw65↗

PubMed2005

Domain reorientation and induced fit upon RNA binding: solution structure and dynamics of ribosomal protein L11 from Thermotoga maritima.

L11, a protein of the large ribosomal subunit, binds to a highly conserved domain of 23S rRNA and mediates ribosomal GTPase activity. Its C-terminal domain is the main determinant for rRNA binding, whereas its N-terminal domain plays only a limited role in RNA binding. The N-terminal domain is thought to be involved in interactions with elongation and release factors as well as with the antibiotics thiostrepton and micrococcin. This report presents the NMR solution structure of the full-length L11 protein from the thermophilic eubacterium Thermotoga maritima in its free form. The structure is based on a large number of orientational restraints derived from residual dipolar couplings in addition to conventional NOE-based restraints. The solution structure of L11 demonstrates that, in contrast to many other multidomain RNA-binding proteins, the relative orientation of the two domains is well defined. This is shown both by heteronuclear 15N-relaxation and residual dipolar-coupling data. Comparison of this NMR structure with the X-ray structure of RNA-bound L11, reveals that binding not only induces a rigidification of a flexible loop in the C-terminal domain, but also a sizeable reorientation of the N-terminal domain. The domain orientation in free L11 shows limited similarity to that of ribosome-bound L11 in complex with elongation factor, EF-G.

Models, Molecular↗