Principles of Bioinorganic Chemistry - 2003

Lecture Date Lecture Topic Reading Problems1 9/4 (Th) Intro; Choice, Uptake, Assembly of Mn+ Ions Ch. 5 Ch. 12 9/ 9 (Tu) Metalloregulation of Gene Expression Ch. 6 Ch. 23 9/11 (Th) Metallochaperones; Metal Folding, X-linkingCh. 7 Ch. 34 9/ 16 (Tu) Zinc Fingers; Metal Folding; Cisplatin Ch. 8 Ch. 45 9/18 (Th) Electron Transfer; Fundamentals Ch. 9 Ch. 56 9/23 (Tu) Long-Distance Electron Transfer Ch. 9 Ch. 67 9/25 (Th) Hydrolytic Enzymes, Zinc, Ni, Co Ch. 10 Ch. 78 9/30 (MU)Model Complexes for Metallohydrolases Ch. 109 10/2 (MU)Dioxygen Carriers: Hb, Mb, Hc, Hr Ch. 1110 10/7 (Tu) O2 Activation, Hydroxylation: MMO, P-450, R2Ch. 11 Ch. 811 10/9 (Th) Model Chemistry for O2 Carriers/Activators Ch. 11 Ch. 912 10/16 (Th) Complex Systems: cyt. oxidase; nitrogenase Ch. 12 Ch. 1013 10/21 (Tu) Metalloneurochemistry/Medicinal Inorg. Chem.Ch. 12 Ch. 1114 10/23 (Th) Term Examination Ch. 12 Ch. 12

Metallochaperones; Metal FoldingPRINCIPLES:

•Metallochaperones guide and protect metals to natural sites•Chaperone and target receptor protein structurally homologous•Metal-mediated protein structure changes affect transcription•Metal-mediated protein structure changes affect translation•Metal-induced protein structure changes also activate enzymes•Metal-induced bending of DNA affects function•Metal ionic radii and M–L water bridging are used to advantage

ILLUSTRATIONS:•Copper insertion into metalloenzymes•Zinc finger proteins control transcription•Ca2+, a second messenger and sentinel at the synapse•Cisplatin, an anticancer drug

Zinc Fingers - Discovery, StructuresA. Klug, sequence gazing, proposed zinc fingers for TFIIIA, which controls the transcription of 5S ribosomal RNA.Zn2+ not removed by EDTA. 9 tandem repeats. 7-11 Zn/protein.Y or F – X – C – X2,4 – C – X3 – F – X5 – L – X2 – H – X3,4 – H – X2,6 CC C H HHHH

The coordination of two S and 2 N atoms from Cys and His residues was supported by EXAFS; Zn–S, 2.3 Å; Zn–N, 2.0 Å. Td geometry.The protein folds only when zinc is bound; > 1% of all genes have zinc finger domains.

X-ray Structure of a Zinc Finger Domain

Structure of a Three Zinc-Finger Domain of Zif 268 Complexed to an Oligonucleotide Containing

its Recognition Sequence

The Specificity of Zinc for Zinc-finger DomainsKd value: 2 pM5nM 2mM3mMMetal ion: Zn2+ Co2+ Ni2+ Fe3+

+ 3/5 Δo

- 2/5 Δo

LFSE = -5(2/5 Δo) + 2(3/5 Δo) = -4/5 Δo+ 2P (small)

= -7440 cm-1 (since Δo = 9300 cm-1) = -21.3 kcal mol-1

For [Co(H2O)6]2+

- 3/5 Δt

+ 2/5 Δt

LFSE = -4(3/5 Δt) + 3(2/5 Δt) = -6/5 Δt+ 2P (small)

= -5880 cm-1 (since Δt = 4900 cm-1) = -16.8 kcal mol-1

For [Co(Cys)2(His)2]

Thus Co2+ loses 4.8 kcal mol-1 in going from aqueous solutionto the zinc finger environment; Zn2+ does not.

Metal Folding of BiopolymersPRINCIPLES:

•Metal-mediated protein structure changes affect transcription•Metal-mediated protein structure changes affect translation•Metal-induced protein structure changes also activate enzymes•Metal-induced DNA structure changes effect anticancer activity

ILLUSTRATIONS:•Ca2+, a second messenger and sentinel at the synapse•Cisplatin, an anticancer drug

Roles of Calcium in Protein Folding and Activation

Calcium at the synapse: binds to the C2 domains of synaptotagmin and helps to control neurological signal transduction. Synaptotagmin is the sensor for Ca2+ influx.Calcium also binds to calmodulin. As Ca2+ levels in the cell increase, calmodulin senses these changes and binds to its target proteins with Kd values of 10 - 100 nM or less. The following are amino acid sequences from activated proteins:

Synaptotagminis a calcium sensor

Synaptotagmin III with Mg2+

C2A-C2B crystal structureBrunger and coworkers 1999

How does Ca2+ activate synaptotagmin?

Dimerizationvia C2

domains?

10-100 mM Ca2+

PhospholipidBinding

mM Ca2+

Binding toproteinpartner

(syntaxin)

Change inConformation?

ElectrostaticSwitch?

Ca2+

C2A C2B

280 340 280 470FRET

C2A C2B

Garcia, R. A.; Forde, C. A.; Godwin, H. A. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5883-5888.

Addition of Calcium Results in anIncrease in Energy Transfer

290 390 490 590wavelength (nm)

⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨

⟨⟨⟨⟨⟨⟨⟨⟨ ⟨⟨⟨⟨⟨

01234567

0 0.01 0.02 0.03Concentraton of Calcium (M)

apo mMCa2+

⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨

⟨⟨⟨⟨ ⟨ ⟨ ⟨ ⟨

⟨ ⟨ ⟨

⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨ ⟨ ⟨ ⟨ ⟨ ⟨ ⟨ ⟨

⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨

⟨ ⟨ ⟨ ⟨ ⟨ ⟨ ⟨

⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨⟨

⟨ ⟨ ⟨ ⟨ ⟨ ⟨ ⟨ ⟨

01234567

0 0.002 0.004 0.006Concentration of Divalent Metal (M)

⟨ Ca2+ ratio⟨ Ba2+ ratio⟨ Mg2+ ratio⟨ Sr2+ratio

Other metal ions that can promote fusionalso confer this conformation change,but to a lesser extent than does Ca2+

Garcia, R. A.; Forde, C. A.; Godwin, H. A. Proc. Natl. Acad. Sci. U.S.A.

2000, 97, 5883-5888.

Calmodulin - Properties and StructureProperties: 148 amino acids; binds up to four Ca2+ ions. Primary structure conserved over many species. Binds calcium in pairs of “EF hand” domains. Calcium coordinated by 3 monodentate Asp, 1 bidentate Glu, 1 peptide backbone carbonyl, and 1 water molecule. Seven-coordination favors Ca2+ over Mg2+.

Ca2+ Kd, 10-6 to < 10-7 M

Mg2+ Kd, ~ 10-3 M

Structural Changes in Calmodulin upon Binding to a Target Peptide Domain

Note how the target peptide is encapsulated following conformational changes in the long central helix

A Fluorescent Sensor for Calcium Binding in Cells

Can be used to correlate with synaptotagmin as well as calmodulin response to Ca2+ levels

•In eukaryotes iron homeostasis is achieved by regulating the concentrations of the transferrin receptor and apoferritin at the translational level. IRP’s are the conformational switch and utilize an Fe4S4 cluster to alter protein structure.

•A mercury resistance operon in bacteria detoxifies R2Hg by lyase and reduction to Hg(0) which volatilizes harmlessly out of the cell. MerR is the sensor which undergoes a conformational change upon Hg binding and activates the genes at the level of transcription. This topic was not covered in class, but please read Chapter 6, Section 6.3.

Summary - Points to Remember

•Zinc fingers form upon metal-binding to the apo peptide domains and activate transcription of thousands of genes. Evidence is accumulating that zinc-binding domains also act as metalloregulators in response to zinc levels in the cell.•Synaptotagmin C2 domains are the calcium sensors at the synapse responsible for Ca-dependent signal transduction in helping to achieve synaptic plasticity.•Calmodulin alters its structure upon calcium influx into cells and activates many genes at the transcriptional level. Calmodulin allows calcium to serve as a second messenger in response to signals at the cell surface, affording a signal transduction pathway.

Summary - Points to Remember, cont’d

What Do These People Have in Common?

Shelley 30 Schumann 39Keats 26 Chopin 39Heine 59 Nietsche 56Emily Brontë 22 Gauguin 55Ann Brontë 29D.H. Lawrence 45George Orwell 47

Bacterial infection

Syphilis

Sir Alexander Fleming (1881 - 1955),a Study in Serendipity

β-lactam ring

N

S

OCO 2H

HNO

R

1922 - While suffering from a cold, Fleming made a culture from his own nasal secretions. As he examined the culture dish, filled with yellow βacteria, a tear fell from his eye into the dish. The next day he found a clear space where the tear had fallen: the tear caused lysis of the βacteria βut was harmless to human tissue. The antiβiotic enzyme responsiβle was named lysozyme, βut was of little practical importance since the βacteria were relatively harmless.

1928 - While working on cultures of influenza grown in petri dishes, Fleming noticed an unusual clear area in one dish. Further examination revealed a βit of mold that had fallen into the dish. Recalling his lysozyme experience, he con-cluded that the mold produced something deadly to the staphylococcus βacteria in the culture dish. Fleming isolated the mold and named the antiβiotic suβstance it produced penicillin.

Penicillin: inhiβits cell wall synthesis in βacteria cells leak H2O and literally explode!

toxic agentpro-drug, Salvarsanthe first "magic bullet"

in vivo

Paul Ehrlich: Discovery of a Treatment for Syphilis

605 organoarsenic compounds screened before # 606 wasdiscovered. Actually a pro-drug:

HO

NH2

As As As

H2N

OHOH O

NH2

1. Random Screening: Folk medicine Sea life: plants, animals - natural product chemistry Activity structure/function relationships

2. Specificity - the Key to Rational Design Chemoreceptor Idea (Paul Ehrlich)

Development of Anticancer Drugs

Questions: Can we design compounds by understanding how the best agents work?Can we harness serendipity by modern combinatorial synthetic methods?

Factors Associated with Antitumor Drug Development

A. Serendipity: > 7000:1 in most screens

B. Perseverance: ~ 13 years between laboratory and pharmacy

C. Cost: > $750 M

D. Moral questions: gene therapy

E. The aversion to metal-based pharmaceuticals

Cisplatin: From DNA Damageto Cancer Therapy

Idea

DiscoveryUnderstanding

& Invention

SerendipityCreativity

Intelligence

Two cis amine ligandsNeutral Pt(II) or Pt(IV) compounds

Two cis leaving groupsH

3

N

P t

H3

N O

O

O

O

P t

C l

C l

H2

N

N

H2

H3

N

H3

N

P t

C l

N H3

C l

Active Inactive

Platinum Antitumor Drugs

The Cisplatin Family

H3

N

P t

C l

C lH3

N

1845 Peyrone's chloride1965 Inhibition of cell growth observed1969 Demonstration of antitumor activity1978 FDA approval for genitourinary tumors2000 Clinical use for many types of cancer

cisplatin

carboplatinoral analogue

NHPt

NH2

ClNH2

H3NPt

CyH2N Cl

ClO2CR

RCO2

0

20

40

60

80

100

0 1 2 3 4 5

1994 overall1970 stage I1970 stage II1970 stage III

Cisplatin: An Extraordinary Anticancer Drug

Years after Orchidectomysource: I. Damjanov, Scientific

American, 1995

% S

urvi

val

•Cisplatin is highly effective against testicular cancer. It is also used to treat head, neck, ovarian, and several other types of human tumors.

Cisplatincis-diamminedichloroplatinum(II)

w/ cisplatin

w/o cisplatin

Pt

H3N Cl

H3N Cl

.

DNA is the Biological Target of Cisplatin

+H3N

PtClH3N

OH2

H3N

H3NH3N

ClH3N

Cl passivediffusion aquation

DNA adductformation PtPt

u Cisplatin diffuses into cells, aquates, and attacks cellular targets, DNA, RNA and proteins.

NN

N

N

NN

O

N

NH

H

O

H

HH

C G7

189

2 3456

1 23

456 N

NN

N

NN

NO

O

H3C

H

HH

AT7

2 3456

18

91 2

345

6

u It is generally accepted that DNA is the main target, with platinum coordinating to N7 of the purine nucleobases guanine and adenine.