4 research outputs found
Characterization of DNA Binding by the Isolated NâTerminal Domain of Vaccinia Virus DNA Topoisomerase IB
Vaccinia
TopIB (vTopIB), a 314-amino acid eukaryal-type IB topoisomerase,
recognizes and transesterifies at the DNA sequence 5âČ-(T/C)ÂCCTTâ,
leading to the formation of a covalent DNAâ(3âČ-phosphotyrosyl<sup>274</sup>)âenzyme intermediate in the supercoil relaxation
reaction. The C-terminal segment of vTopIB (amino acids 81â314),
which engages the DNA minor groove at the scissile phosphodiester,
comprises an autonomous catalytic domain that retains cleavage specificity,
albeit with a cleavage site affinity lower than that of the full-length
enzyme. The N-terminal domain (amino acids 1â80) engages the
major groove on the DNA face opposite the scissile phosphodiester.
Whereas DNA contacts of the N-terminal domain have been implicated
in the DNA site affinity of vTopIB, it was not known whether the N-terminal
domain per se could bind DNA. Here, using isothermal titration calorimetry,
we demonstrate the ability of the isolated N-terminal domain to bind
a CCCTT-containing 24-mer duplex with an apparent affinity that is
âŒ2.2-fold higher than that for an otherwise identical duplex
in which the pentapyrimidine sequence is changed to ACGTG. Analyses
of the interactions of the isolated N-terminal domain with duplex
DNA via solution nuclear magnetic resonance methods are consistent
with its DNA contacts observed in DNA-bound crystal structures of
full-length vTopIB. The chemical shift perturbations and changes in
hydrodynamic properties triggered by CCCTT DNA versus non-CCCTT DNA
suggest differences in DNA binding dynamics. The importance of key
N-terminal domain contacts in the context of full-length vTopIB is
underscored by assessing the effects of double-alanine mutations on
DNA transesterification and its sensitivity to ionic strength
Sequential Protein Expression and Capsid Assembly in Cell: Toward the Study of Multiprotein Viral Capsids Using Solid-State Nuclear Magnetic Resonance Techniques
While
solid-state nuclear magnetic resonance (ssNMR) has emerged
as a powerful technique for studying viral capsids, current studies
are limited to capsids formed from single proteins or single polyproteins.
The ability to selectively label individual protein components within
multiprotein viral capsids and the resulting spectral simplification
will facilitate the extension of ssNMR techniques to complex viruses. <i>In vitro</i> capsid assembly by combining individually purified,
labeled, and unlabeled components in NMR quantities is not a viable
option for most viruses. To overcome this barrier, we present a method
that utilizes sequential protein expression and in cell assembly of
component-specifically labeled viral capsids in amounts suitable for
NMR studies. We apply this approach to purify capsids of bacteriophage
Ï6 isotopically labeled on only one of its four constituent
protein components, the NTPase P4. Using P4-labeled Ï6 capsids
and the sensitivity enhancement provided by dynamic nuclear polarization,
we illustrate the utility of this method to enable ssNMR studies of
complex viruses
Docking Interactions of Hematopoietic Tyrosine Phosphatase with MAP Kinases ERK2 and p38α
Hematopoietic tyrosine phosphatase (HePTP) regulates
orthogonal
MAP kinase signaling cascades by dephosphorylating both extracellular
signal-regulated kinase (ERK) and p38. HePTP recognizes a docking
site (D-recruitment site, DRS) on its targets using a conserved N-terminal
sequence motif (D-motif). Using solution nuclear magnetic resonance
spectroscopy and isothermal titration calorimetry, we compare, for
the first time, the docking interactions of HePTP with ERK2 and p38α.
Our results demonstrate that ERK2âHePTP interactions primarily
involve the D-motif, while a contiguous region called the kinase specificity
motif also plays a key role in p38뱉HePTP interactions.
D-MotifâDRS interactions for the two kinases, while similar
overall, do show some specific differences
Structure of the CâTerminal Helical Repeat Domain of Eukaryotic Elongation Factor 2 Kinase
Eukaryotic
elongation factor 2 kinase (eEF-2K) phosphorylates its
only known physiological substrate, elongation factor 2 (eEF-2), which
reduces the affinity of eEF-2 for the ribosome and results in an overall
reduction in protein translation rates. The C-terminal region of eEF-2K,
which is predicted to contain several SEL-1-like helical repeats (SLRs),
is required for the phosphorylation of eEF-2. Using solution nuclear
magnetic resonance methodology, we have determined the structure of
a 99-residue fragment from the extreme C-terminus of eEF-2K (eEF-2K<sub>627â725</sub>) that encompasses a region previously suggested
to be essential for eEF-2 phosphorylation. eEF-2K<sub>627â725</sub> contains four helices, of which the first (αI) is flexible,
and does not pack stably against the ordered helical core formed by
the last three helices (αIIâαIV). The helical core
is structurally similar to members of the tetratricopeptide repeat
(TPR) family that includes SLRs. The two penultimate helices, αII
and αIII, comprise the TPR, and the last helix, αIV, appears
to have a capping function. The eEF-2K<sub>627â725</sub> structure
illustrates that the C-terminal deletion that was shown to abolish
eEF-2 phosphorylation does so by destabilizing αIV and, therefore,
the helical core. Indeed, mutation of two conserved C-terminal tyrosines
(Y712A/Y713A) in eEF-2K previously shown to abolish eEF-2 phosphorylation
leads to the unfolding of eEF-2K<sub>627â725</sub>. Preliminary
functional analyses indicate that neither a peptide encoding a region
deemed crucial for eEF-2 binding nor isolated eEF-2K<sub>627â725</sub> inhibits eEF-2 phosphorylation by full-length eEF-2K. Taken together,
our data suggest that the extreme C-terminal region of eEF-2K, in
isolation, does not provide a primary docking site for eEF-2