Transcript Metal Regulation and Signalling - Zn Proteins

Metal Ion Receptors and Signaling
BIG area
context:
bioinorganic research moves beyond metalloenzymes to more
subtle roles for metals: structural roles
Why? X-ray crystallography.
Metal Ion Receptors and Signaling
What do the metal ions do?
Metal ions stabilize folded protein structure.
And limit possible number of conformations,
 make the correct structure.
Typically metal is in a buried site.
Especially used intracellularly due to reducing
environment which precludes using disulfide bonds
to enforce protein fold.
Metalloregulatory Proteins
Typically, these proteins exist in two distinct interconvertable
conformations, with and without M.
Binding of M favors one and effectively can act as a switch to signal some
change or consequence.
Structural metalloproteins overwhelmingly use zinc.
Why Zn(2+) ???
Why Zn(2+) ???
1. Borderline acid character: binds variety of ligands, Cys, His, Asp
2. CFSE = 0
Flexible coordination sphere: Td, Oh, sq. pyr, trig. Bpy.
3. No redox; Non-toxic
4. Abundant, high concentration (almost mMolar)
Structural Zn(2+) Domains
Prototype Zn “finger” domains called ‘CCHH’ for Cys (XX) Cys (XX) His (XX) His.
May have multiple Zn ions in multiple “fingers”, where Zn … Zn is > 26 Å.
Other dinuclear Zn domains also observed where Zn – Zn distance 13-18 Å
LIM, RING, GATA, CRD (cys-rich domain), nuclear hormone receptor motif
Which type of Zn domain is present depends on species.
Structural Zn proteins have different functions, but all create interfaces for
macromolecular interactions: protein-protein, protein-DNA, protein-RNA,
protein-polysaccharides.
No Zn binding motifs found in the e. coli genome, in contrast to an abundance of
motifs in eukaryotic cells. Speculation that higher organisms developed better
means of Zn retention?
Zn(2+) sites
Typically is a distorted Td so CN = 4
This serves to constrain protein conformation by binding at four different aa’s
Typically 2 or more Cys- ligands, where frequency is Cys > His >> Asp, Glu
Binds at either e or d N of His (what does this mean?)
and other His NH group H-bonds to O atom of water or amino acid
Studies on Zn(2+) domains
Zn(2+) is spectroscopically silent (what does this mean?)
What could be substituted for Zn?
Co(2+) is similar size and has similar L preferences.
So Co replaces Zn easily.
Co(2+) in a Td site: what do you recall?
Jeremy Berg: first Zn protein “model” work using consensus polypeptide (CP)
sequences: CP-CCHH, CP-CCHC, CP-CCCC and substituted in Co(II).
Could observe spectroscopically the increase in Cys coordination causing main
~640 nm to shift to longer wavelengths.
Could also use Co substitution to explore thermodynamics.
Titrate in Co to get Kb(Co); titrate back in Zn, get Kb(Zn)
Zn finger protein function and structure
Zn finger creates interfaces for protein- nucleic acid interactions,
acts as transcriptional activators or repressors
Classic structural motif uses two antiparallel beta sheets followed by
alpha helix, to create a hydrophobic core.
Zn coordinated by 2 Cys in hairpin turn and 2 His in helix.
(PDB 1ZAA)
Zn finger protein structure
Dinuclear Zn protein sites
Two sets of donor atoms, Zn-Zn about 13-18 Å apart, so NOT a dimer
LIM domain acts as a specific protein-protein interface.
LIM-domain proteins function to assemble multiple components into
macromolecular complexes. Involved in cell differentiation and growth.
Dinuclear Zn protein sites
Nuclear hormone receptor proteins bind DNA with ZnCys4 sites.
Dinuclear Zn protein sites
CRM (cysteine rich domain) motif involved in signal transduction proteins such as
kinases.
One Zn bound by CxxC and HxxC regions in two loops.
Dinuclear Zn protein sites
Zn-Zn = 14.2 Å apart
Has triple antiparallel beta sheets.
RING motif proteins have variety of functions, regulation of gene expression,
DNA repair, recombination and peroxisomal assembly. (?)
Metal ion signaling
Best understood for Ca2+
Zn2+ examples similar in that responds to changes in [Zn]
Zn in neuromodulation where Zn ions released from synaptic vesicles then bind
to post-synaptic receptors.
Zn promotes protein oligomerization of human growth hormone.
Metalloregulatory Proteins
Transduce metal ion signals into gene or protein expression
Function in metal ion homeostasis and detoxification
Identified regulators for Zn, and also Cu and Fe in yeast.
Deficiency of M activates genes for ‘components’ for M uptake.
Example of Cu-mediated gene activation for metallothionein.
There is a Cu sensor protein Ace1, and Amt1 mediates the expression of
metallothionein genes. Ace1 and Amt1 are bridged by a two domain protein
with Zn structural in one domain and a Cu4 cluster in other domain, whose
conformation controlled by Cu(I) binding.
Escherichia coli:
a cross-section of a small portion of a cell.
Green: the cell wall, with two concentric
membranes studded with transmembrane
proteins. A large flagellar motor crosses
the entire wall, turning the flagellum that
extends upwards from the surface.
Blue & purple : the cytoplasmic area.
Purple molecules are ribosomes and the
small, L-shaped maroon molecules are
tRNA, and the white strands are mRNA.
Enzymes are shown in blue.
Yellow & orange: the nucleoid, with the
long DNA circle shown in yellow, wrapped
around HU protein (bacterial
nucleosomes). In the center of the
nucleoid region shown here, you might
find a replication fork, with DNA
polymerase (in red-orange)
replicating new DNA.
© David S. Goodsell 1999.
Associate Professor of Molecular
Biology
Department of Integrative Structural
and Computational Biology
Scripps Research Institute
The crowded cell