How metal ions control protein structure and dynamics

(and biomolecular interactions)A: Calmodulin

B: Zinc fingers

Calcium-binding proteins:

Calcium regulates proteinstructure, dynamics and interactions

The EF hand motif

Lewit-Bentley, A. & Réty, S. (2000)Curr. Op. Struct. Biol., 10, 637 - 643

Numerous Ca-binding proteins contain pairs of this motif

Labels E and F stem from parvalbumin, where this motif was first discovered

http://www.agr.nagoya-u.ac.jp/~mcr/Research/EF-hand.html

Calmodulin

• Monomeric, 148 aa, 17 kDa• Binds up to 4 Ca2+

• 2 x 2 EF-hands• Why specific for Ca2+ ?

• log K ≈ 6-7 (Mg2+: 3-4)

•7 oxygen ligands (hard !): –Not suitable for soft/borderline ions (Cu(I/II), Zn(II))–Mg2+ or Fe3+ too small

Calmodulin

1 EF hand

A pair of EF hands Full-length Ca4-calmodulin

X-ray: pdb 1cll; Chattopadhyaya, R.,  Meador, W.E.,  Means, A.R.,  Quiocho, F.A. J. Mol. Biol. 228, 1177-1192 (1992)

Two domains connected by long-helix

Apo CaM: Disruption of central helix

• NMR solution structure

• No fixed orientation of the two domains w.r. to each other

• Key point: Ca2+

binding changes protein structure and dynamics

NMR structure: pdb 1dmo; M Zhang, T Tanaka, M Ikura:Calcium-induced conformational transition revealed by the solution structure of apo calmodulin. Nature structural biology. (1995) 2, pp. 758-67

Cooperativity• Usually, simple

binding curves are hyperbolical

• Cooperativity is frequent in biological systems

• Binding curves are sigmoidal

• Means: Binding of the first Ca enhances binding of the second and so on

• Will see again when discussing Hb

http://ead.univ-angers.fr/~jaspard/Page2/COURS/7RelStructFonction/2Biochimie/5Signalisation/2Calmoduline/1Calmodulin.htm

Bo

un

d C

a2+

non-cooperative binding

cooperative binding in CaM

Ca-loaded CaM interacts with a plethora of proteins

• Strong interaction (Kd 10-100 nM) with many different proteins: Strange

How ?

• Ca2+ binding induces the formation of hydrophobic patches on calmodulin surface

• CaM-binding proteins have a positively charged amphipathic helix (hydrophobic and hydrophilic sides)

• Interacts with both hydrophobic patches

calmodulin “mitts”

WE Meador, AR Means, FA Quiocho: Target enzyme recognition by calmodulin: 2.4 A structure of a calmodulin-peptide complex. Science. (1992) 257, 1251-5

Less schematic:

Summary

• Binding of Ca2+ to calmodulin changes protein structure and dynamics

• This change in properties enables CaM to bind to a plethora of other proteins, but only in the presence of Ca2+ (and only Ca2+)

• This is how, by regulation of intracellular Ca2+

concentrations, the activity of many other proteins can be regulated

Bio-Inorganic ChemistryLecture 6b

Zinc fingers:

Control of protein structure and biomolecular interactions

Zinc proteins

Zinc enzymesZinc transporting proteins

Enzymes in which zinc modulates activity

Zinc fingers and other proteins with structural zinc

Hydrolasesothers

peptidases

nucleases

Metallo--lactamases

others

Zinc fingers

• Small protein domains

• Classical: C2H2 ligand set

• Tetrahedral coordination• Unfolded without zinc• DNA binding (only in

presence of zinc)

x xx xx xx xx xx xC H

x \ / xx Zn xx / \ xC H

x x x x x x x x x x

x xx xx xx xx xx xC H

x \ / xx Zn xx / \ xC H

x x x x x x x x x x

N

C

• Transcription factors• Often occur in multiples

Zif268 binding to DNA

M Elrod-Erickson, TE Benson, CO Pabo

3 fingers in one proteinInteraction with the major groove

DNA recognition: a code ?

Corbi, Nicoletta; Libri, Valentina; Onori, Annalisa; Passananti, Claudio. Biochemistry and Cell Biology (2004), 82(4), 428-436.

Interactions are mediated by H-bonds in major groove

His (+3)

Arg (-1)

Asp (+2)

G

G

C

Artificial zinc fingers

• Based on unravelling the details of zinc finger DNA recognition

• Re-design zinc finger proteins for recognition of other DNA sequences

• Promising for medical applications: e.g. Gene regulation/targeting, antiviral therapy

Today: 116 different zinc finger families Family Description

DUF537 Protein of unknown function, DUF537

FLYWCH FLYWCH zinc finger domain

FYVE FYVE zinc finger

GATA GATA zinc finger

KRAB KRAB box

SWIM SWIM zinc finger

U-box U-box domain

zf-CCCH Zinc finger C-x8-C-x5-C-x3-H type (and similar)

zf-CCHC Zinc knuckle

zf-CXXC CXXC zinc finger

zf-HIT HIT zinc finger

zf-Sec23_Sec24 Sec23/Sec24 zinc finger

ADK_lid Adenylate kinase, active site lid

AKAP95 A-kinase anchoring protein 95 (AKAP95)

BAF1_ABF1 BAF1 / ABF1 chromatin reorganising factor

DNA_ligase_ZBD

NAD-dependent DNA ligase C4 zinc finger domain

C1_4 TFIIH C1-like domain

DUF500 Family of unknown function (DUF500)

DUF629 Protein of unknown function (DUF629)

Ribosomal_S27 Ribosomal protein S27a

RpoE2 Archaeal DNA-directed RNA polymerase subunit E'' (RpoE'' or RpoE2)

zf-A20 A20-like zinc finger

zf-AN1 AN1-like Zinc finger

zf-C4 Zinc finger, C4 type (two domains)

Family Description

DUF537 Protein of unknown function, DUF537

FLYWCH FLYWCH zinc finger domain

FYVE FYVE zinc finger

GATA GATA zinc finger

KRAB KRAB box

SWIM SWIM zinc finger

U-box U-box domain

zf-CCCH Zinc finger C-x8-C-x5-C-x3-H type (and similar)

zf-CCHC Zinc knuckle

zf-CXXC CXXC zinc finger

zf-HIT HIT zinc finger

zf-Sec23_Sec24 Sec23/Sec24 zinc finger

ADK_lid Adenylate kinase, active site lid

AKAP95 A-kinase anchoring protein 95 (AKAP95)

BAF1_ABF1 BAF1 / ABF1 chromatin reorganising factor

DNA_ligase_ZBD

NAD-dependent DNA ligase C4 zinc finger domain

C1_4 TFIIH C1-like domain

DUF500 Family of unknown function (DUF500)

DUF629 Protein of unknown function (DUF629)

Ribosomal_S27 Ribosomal protein S27a

RpoE2 Archaeal DNA-directed RNA polymerase subunit E'' (RpoE'' or RpoE2)

zf-A20 A20-like zinc finger

zf-AN1 AN1-like Zinc finger

zf-C4 Zinc finger, C4 type (two domains)

zf- Topoisomerase DNA binding C4 zinc finger

zf-piccolo Piccolo Zn-finger

zf-nanos Nanos RNA binding domain

zf-B_box B-box zinc finger

zf-CHY CHY zinc finger

zf-CSL CSL zinc finger

Baculo_ME53 Baculoviridae ME53

zf-C2HC5 Putative zinc finger motif, C2HC5-type

zf-DHHC DHHC zinc finger domain

zf-C3HC4 Zinc finger, C3HC4 type (RING finger)

zf-MYM MYM-type Zinc finger

zf-C4_ClpX ClpX C4-type zinc finger

zf-FPG_IleRS Zinc finger found in FPG and IleRS

zf-GRF GRF zinc finger

zf-LSD1 LSD1 zinc finger

Transposase_35 Putative transposase DNA-binding domain

eIF-5_eIF-2B Domain found in IF2B/IF5

zf-dskA_traR Prokaryotic dksA/traR C4-type zinc finger

zf-U1 U1 zinc finger

zf-CW CW-type Zinc Finger

zf-HYPF HypF finger

zf-BED BED zinc finger

PRE_C2HC Associated with zinc fingers

zf-DBF DBF zinc finger

zf-AD Zinc-finger associated domain (zf-AD)

zf-C2H2 Zinc finger, C2H2 type

zf-C3HC C3HC zinc finger-like

zf-RNPHF RNPHF zinc finger

zf-MIZ MIZ zinc finger

Hep_core_N Hepatitis core protein, putative zinc finger

HCV_NS5a_1a Hepatitis C virus non-structural 5a zinc finger domain

MATH MATH domain

PHD PHD-finger

R3H R3H domain

SCAN SCAN domain

SWIRM SWIRM domain

WRKY WRKY DNA -binding domain

zf-UBP Zn-finger in ubiquitin-hydrolases and other protein

Androgen_recep Androgen receptor

CBFD_NFYB_HMF

Histone-like transcription factor (CBF/NF-Y) and archaeal histone

DAGK_cat Diacylglycerol kinase catalytic domain (presumed)

Fork_head Fork head domain

Gene66 Gene 66 (IR5) protein

RecR RecR protein

Ribosomal_L37ae

Ribosomal L37ae protein family

Ribosomal_S27e

Ribosomal protein S27

RNA_POL_M_15KD

RNA polymerases M/15 Kd subunit

WT1 Wilm's tumour protein

XPA_C XPA protein C-terminus

BTK BTK motif

GCR Glucocorticoid receptor

THAP THAP domain

HypA Hydrogenase expression/synthesis hypA family

PDCD2_C Programmed cell death protein 2, C-terminal putative domain

Prog_receptor Progesterone receptor

IBR IBR domain

STE STE like transcription factor

zf-RanBP Zn-finger in Ran binding protein and others

zf-MYND MYND finger

CCT CCT motif

Sec23_helical Sec23/Sec24 helical domain

Sec23_trunk Sec23/Sec24 trunk domain

SIR2 Sir2 family

NHL NHL repeat

DZF DZF

TAFH NHR1 homology to TAF

Zinc fingers are amongst the most populous domains in the human genome

The zinc fingers

• Can have His2Cys2, Cys3His, or Cys4 ligand set

• Can have 1 or 2 zinc sites• Can be classified by the pattern of ligands in

the sequence• E.g. Pattern for GATA-type zinc fingers:• C - x - [DN] - C - x(4,5) - [ST] - x(2) - W - [HR] -

[RK] - x(3) - [GN] - x(3,4) - C - N - [AS] - C

Some principles in DNA-binding zinc fingers

• Isolated fingers, but more than one required (can be 2-9, or even more)

• 2 zinc working together to structure protein

• Directly bridged by 2 Cys sulfurs

Zinc fingers, zinc clusters, and zinc twists in DNA-binding protein domains, BERT L. VALLEE, JOSEPH E. COLEMAN, AND DAVID S. AULD, PNAS 88, 999-1003 (1991).

HIV nucleocapsid zinc fingers

• Small, basic, 2 zinc fingers• Nucleic acid chaperone: protects viral

RNA and is important for reverse transcription (RNA as template for DNA synthesis)

• Drugs targeting the zinc fingers are in development: e.g. 2,2'-dithiobisbenzamides (DIBAs), azadicarbonamide (ADA))

http://jlevinlab.nichd.nih.gov/research.html

TL South, MF Summers:Protein science (1993) 2, pp. 3-19

CCHC ligand set

Summary

• Zinc fingers are important for mediation of bio-molecular interactions

• Cys2His2, Cys3His, Cys4 coordination modes

• Unfolded without zinc