Validation of Copper PDB files

Analysis of Copper inPDB filesJoana PintoJuly 2009

ObjectiveAnalyse the structures of Copper containing proteins and look for errors

Keep making protein structures more and more accurateRelying on improved software and better knowledge of protein structure

PDB Files ErrorsDifficulty in distinguish certain atoms

Errors are kept because its not a curated database.

3Ions in ProteinsIron, Zinc, Calcium, Magnesium, Chlorine or Iodine, etc.

CopperTwo stable oxidation states[Ar] s1d10Copper proteins classification

4Type I Copper ProteinsBlue Proteins

2 Histidines, 1 Cysteine and 1 Methionine

There are about 50.000 proteins in this familyCuN - HistidineN - HistidineS - MethionineCysteine - S5Type I Copper Proteins - Plastocyanin

6Type II Copper Proteins3 Histidines + Histidine or H2O or Methionine or Cysteine

Colorless

CuN - HistidineN - HistidineN - HistidineX7Type II Copper Proteins - PHMPeptidylglycine -Hydroxylating Monooxygenase

8Type II Copper Proteins - PHM

CuACuB CuA center is coordinated by three Histidine residues and a water molecule (not visible) whereas CuB is coordinated by two Histidines, a Methionine residue and also a water molecule The two Copper centers are at a distance of about 11 from each other.

9Type III Copper Proteins Binuclear centers: 3 Histidines and O2

CuN - HistidineN - HistidineN - HistidineCuON - HistidineN - HistidineN - HistidineO binuclear centers in which each of the two copper ions CuA and CuB - are coordinated by three nitrogens from three Histidine residues and a Oxygen molecule

10Type III Copper Proteins - Hemocyanin

*Deoxygenated state 4,6 *Oxygenated state 3,6

* Not validated by usHc can be compared to Hb since they both have an oxygen transport function, the main difference is that Hb has a iron-based active site, while Hc has a copper-based active site. Hc is often found in mollusks and arthropodsThe copper ions are found in the N-terminal domain, which is composed by several helices.11Cu+ versus Cu2+Reduced form - cuprousOxidized form - cupric[Ar] 4s03d10Soft ion Soft ligands (S/P)C.N: 2, 3 and 4[Ar] 4s03d9 Hard ion Hard ligands (N/O)C.N: 4, 5 and 6

Comment the fact that, due to the fact of the ions being in constant state transition, the copper is at the same time binding to soft and hard ions.hard: N and OSoft: S and P

12Materials and MethodsPDB Files:

X-ray CrystallographyResolution better than 2.0

Cu+Cu2+Total Files114562Cu+Cu2+Used Files3532113Materials and MethodsWHAT IF: CalculationsSKPNOOGRSION

Distances plot

YASARA:Pictures

14Results Analysis for Cu+ and Cu2+Cu+Total Files114Files Used 35Num of ligandsNum of Copper Ions311437529GeometryNum of Copper IonsTetrahedral24Other13LigandsNum of Copper Ions2 His , 1 Met , 1 Cys 144 His7Others3Cu2+Total Files562Files Used 321Num of ligandsNum of Copper Ions34543845231GeometryNum of Copper IonsTetrahedral331Square Planar8Other45LigandsNum of Copper Ions2 His , 1 Met , 1 Cys 2103 His , 1 H2O69Others5215Results Regular tetrahedrals for Cu+ and Cu2+GeometryNum of Copper IonsTetrahedral24Regular tetrahedral14LigandsNum of Copper Ions2 His , 1 Met , 1 Cys 133 Met, 1 SCN1GeometryNum of Copper IonsTetrahedral331Regular tetrahedral215LigandsNum of Copper Ions2 His , 1 Met , 1 Cys 1493 His, 1 H2O43Others2313 Cu+ ions (spread over 8 PDB files) and 149 Cu2+ ions (spread over 94 PDB files)

distances plot16Results Distances plotAverage positions for allthe intervenient residues in the regular tetrahedrals

difference between the averagepositions is of 0.6 , being this value close to the error expected in the X-ray structure determination

17Results Flipping HistidinesWhen superposed, 5PCY* and 6PCY* show no differences in its structures besides:

a small change in a Proline residue, and a rotation of an Imidazole ring by 180 about C - C.

* Guss, J.M., Harrowell, P.R., Murata, M., Norris, V.A., Freeman, H.C. (1986). Crystal Structure Analysis Of Reduced (Cu I) Poplar Plastocyanin At Six pH Values. J.Mol. Biol 192 (1986): 361-38718Results Flipping HistidinesNo reason for the flipping of the Histidine

At a resolution of both 5PCY (1.80 ) and 6PCY (1.90 ) the Pro puckering tends to be unobservable

5PCY6PCY19ConclusionsToo few data

Coordination Numbers: For Cu+ and Cu2+: most often seen C.N. is 4, 5 and 3.Contradicting some older studies*

No differences between the structures of Cu+ and Cu2+Contradicting some older studies**

*Belle, C., Rammal, W., Pierre, J.L., (2005). Sulfur Ligation in Copper Enzymes and Models. Journal of Inorganic Biochemistry 99: 1929-1936**Taylor, M.K., Stevenson, D.E., Berlouis, L.E.A., Kennedy, A.R., Reglinski J. (2006). Modeling the Impact of Geometric Parameters On The Redox Potential Of Blue Copper Proteins. Journal of Inorganic Biochemistry 100: 250-259if there were substantially more data to analyze, the conclusions would be much more reliable.

20ConclusionsFor coppers with C.N. 4, the most common geometry is tetrahedral

Copper proteins classifications should be revised, for example, Type II classification is ambiguous.

Some older publications should be reviewed 21Future WorkWhat distinguishes between both oxidation states for copper?

Are the differences too small to be detected?

Are there any differences at all?

One of the possibilities to be studied would be the protonation states for Histidine and Cysteine or other factors like protein bondThank You

Questions?