BIG area Metal Ion Receptors and Signaling 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.

The crowded cell 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