title: Structural and functional studies on the human DEAD-box helicase DDX1
creator: Kellner, Julian
subject: 000
subject: 000 Generalities, Science
subject: 570
subject: 570 Life sciences
description: RNA helicases are essential in all steps of RNA maturation, beginning with transcription and ending with RNA decay. They catalyze the separation of nucleic acid double strands and thereby facilitate structural remodeling. Their cellular importance is reflected in severe diseases caused by RNA helicase dysregulation. The largest group of RNA helicases is confined by the so called DEAD-box helicases of superfamily 2, characterized by the signature sequence D-E-A-D. DEAD-box helicases share a structurally conserved core of two RecA-like domains that carry signature motifs involved in ATP-binding, ATP-hydrolysis, RNA-binding and RNA-remodeling. An exceptional member of the DEAD-box protein family is the human RNA helicase DDX1 (DEAD-box helicase 1). In contrast to all other family members, DDX1 harbors a SPRY domain insertion in between the signature motifs of the helicase core.  DDX1 is involved in a plethora of different RNA maturation processes and has been associated with tumor progression. Moreover due to its versatile function in RNA processing, it is hijacked by viruses for their replication. This medical relevance makes DDX1 a potential target for the development of pharmaceutics; however, such an approach would require mechanistic insights into DDX1 function.   This thesis therefore aimed at the structural and functional characterization of DDX1. The structure of the SPRY domain was determined to near atomic resolution. This structure showed two layers of concave shaped, anti-parallel β-sheets that stack onto each other. A comparison with structures of previously described SPRY domains revealed that the general fold is conserved, but the loops that mediate the interaction with partner proteins in other SPRY structures show distinct conformations and sequences in human DDX1-SPRY. A patch of positive surface charge is found in proximity of these interaction loops that is conserved within DDX1 SPRY domains and may replace the canonical protein-protein interaction surface. Based on its orientation in a homology model of DDX1, the SPRY domain possibly also enlarges the RNA binding site of the helicase core. The structural analysis is complemented by a detailed biochemical and biophysical characterization of DDX1 including the SPRY domain. Equilibrium titrations and transient kinetics with fluorescent nucleotide analogs were used to determine ATP- and ADP-binding affinities. The binding of ADP is unexpectedly tight and ADP-affinity is one of the tightest observed for DEAD-box proteins. DDX1 binds ADP tighter by a factor of almost 1000, when compared to its ATP-affinity; the latter is in the range of what has been reported for other helicases. Thus, the enzyme would be arrested in an inactive ADP-bound conformation under physiological conditions. These observations suggest that a nucleotide exchange factor – as described for the DEAD-box protein DDX19 – is necessary to resolve this ADP-stalling dilemma. Furthermore, the affinity for RNA and the influence of RNA on ATP/ADP binding was determined. Analysis of the data revealed synergistic effects of RNA and ATP binding, which were also reflected in RNA stimulated ATP hydrolysis. These observations concluded in a working model for DDX1, in which both, RNA and ATP per se have the ability to induce a conformational change to an “active” state of the helicase.  In conclusion this thesis provides the first structural information on DDX1 and by presenting the SPRY domain structure, it gives insights into the unique protein-protein interaction domain insertion. The biochemical data provide mechanistic details, show an unsual tight ADP binding and cooperativity in ATP and RNA binding, and allowed developing a model for DDX1 substrate binding.
date: 2014
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/17150/1/thesis_for_uni-bib.pdf
identifier: DOI:10.11588/heidok.00017150
identifier: urn:nbn:de:bsz:16-heidok-171502
identifier:   Kellner, Julian  (2014) Structural and functional studies on the human DEAD-box helicase DDX1.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/17150/
relation: ME3135/1-2
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng