Computational Analysis of

Guanine-binding Surfaces on Proteins

 

Zhou Zhu, Dept. of Chemistry, Saint Mary's College

A. E. Hodel, Dept. of Biochemistry, Emory University

 

 

Goal

Materials

Methods

Results

Conclusions

Reference

Goal

    Have you ever wondered how proteins recognize respective ligands without the help of any sophisticated organs such as eyes, noses, or ears?  It is the magic of their complex tertiary structure, whose properties usually determine if the interaction with a particular substrate can take place or not.  This leads us to another question: What are these properties then? 
 
    During the past ten weeks, we have been trying to approach the answer through the study of guanine-binding proteins.  We aim to quantify their binding site by developing numerical templates which describe the protein environment around guanine surfaces.  It will enable us to compare different protein structures and look for patterns in protein-ligand interaction. 

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Materials

    The raw materials that we base our templates on are 84 guanine-binding proteins located in the Protein Data Base (PDB).  They all contain one form of guanine moiety or another and most of them play important roles in biological systems through specific recognition of  various ligands.  For instance, a subclass of them, Ras proteins, are involved in signal transduction from growth factor receptors on the plasma membrane to gene activations within the nucleus.  The “mysterious” switch that turns them on or off is GDP-GTP exchange.  In their active state, ras bind guanosine triphosphate; they are deactivated when GAPs (GTPase activating proteins) simulate the hydrolysis of  GTP to GDP.  The process is reversible by the interaction with certain receptors.
 
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 Methods

    We started from defining a plain guanine surface with 440 vertices (Figure-1 and Figure-2).  For each vertex, twenty-seven properties were calculated.  Twenty of them are amino acid properties, i.e. the shortest distance to every kind of ?-amino acids (alanine, arginine, asparagine, etc).  Six are atom type properties, i.e. the shortest distance to aliphatic carbon, aromatic carbon, basic nitrogen, neutral nitrogen, acidic oxygen and neutral oxygen respectively.  One is self-property, i.e. the shortest distance to the non-guanine part of their ligands. We performed data reduction by transforming the distance arrays to density arrays with the function y=e^-(x*0.9)^4.  The purpose of doing so is to flag those parts touching the surface with density of 1 and those very far away from the surface with density of 0.  Moreover, it allows a smooth transition in between (Figure-3).  To avoid the overweighing of a particular kind of protein, we ran a sequence homology analysis on our 84 proteins and came up with 34 distinct (over 10 amino acid difference) structures.  A list of them is shown in Figure-4.  These individual templates were compared to each other for similarities in the binding environment.  We tried to see how they correlate with the evolutionary relationship of Guanine-binding proteins, which is suggested by their folding tree.  We also made average templates out of them for seeking general pattern. The major computer programs employed in our study are GRASP, MATHEMATICA and MIDAS.  We obtained the folding tree by searching the DALI domain database.

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Results

    We didn’t see any specific recognition motif in terms of particular residue/ligand interactions.  No residue is conserved among all the 34 non-homology structures studied.  However, there is some common general pattern shared by them:

• (i) High density of basic nitrogen around the O6 part of the guanine;
• (ii) High density of acidic oxygen around the N1 and C2 amino part of the guanine;
• (iii) High density of aliphatic carbons everywhere, especially concentrated around the O6 part of the guanine;
• (iv) Relatively low density of aromatic carbon; When they appear, they usually show around the middle flat part of the guanine;
• (v) Heavy shielding by the non-guanine part of the ligand, except at its O6 part;
• (vi) Relatively high density of aspartic acid around N1 and C2 amino part of the guanine.

    Some graphs have been made to display our results visually (Figure-5 and Figure-6).  Due to the limitation of the space on this poster, we will take three types of proteins as the representative of  all 34 distinct ones.  They are Ras, RNAse T1, and DMSO reductase, all with quite different functions and foldings from each other. We detected certain similarity in ligand template and evolutionary relationship by comparing the folding tree (Figure-7) and our binding-surface trees (Figure-8).  Two big families, agreed by both trees, turn out to be one made up of  RNAase, DNA polymerase, Hgprtase and the other made up of Adp-ribosylation factor 1, Ras, Rap, Transducin, Gi Alpha–1, Elongation factor.

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 Conclusions

    The high density of basic nitrogen and acidic oxygen is obviously related to their capacity for hydrogen bonding.  Since neutral ones fail to show such trend, we may suggest that hydrogen bonding is stronger between charged species than uncharged ones as the result of greater dipole moment.  The shape of aromatic residues determines its preference for flat surface in order to achieve maximum interaction.  We don’t have good explanation why aspartic acid is favored over glutamic acid around a basic edge yet.  Maybe this has something to do with the lower entropic cost of holding aspartic acid still.  Its side chain is one-carbon shorter and thus results in less overall entropy of the residue.
 
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Reference 
 
    1. Grasp (Graphical Representation and Analysis of Structural Properties): Anthony Nicholls
    2. Midas (Molecular Display and Simulation System): UCSF
    3. Mathematica: Stephen Wolfram

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Figures
 
 

Figure-1: A plain guanine surface.  It is the surface surrounding guanine base (the double ring shown inside).	Figure-2: A guanine surface with 440 vertices.  The vertices are evenly distributed throughout the entire surface.

 
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Figure-3: Our reduction function: y=e^-(x*0.9)^4.  We used this function to transfer distance arrays into density arrays. It enables us to flag those parts touching the surface with density of 1 and those very far away from the surface with density of 0.  Moreover, it allows a smooth transition in between.

 
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1 Ribonuclease T1	18 DNA Polymerase (1)
2 Ribonuclease Sa	19 DNA Polymerase (2)
3 Ribonuclease Ms	20 mRNA Capping Enzyme (1)
4 Ribonuclease F1	21 mRNA Capping Enzyme (2)
5 Ribonuclease A	22 Human Adp-Ribosylation Factor 1
6 Ras	23 Rat Adp-Ribosylation Factor 1
7 Rap2a	24 Gi Alpha 1
8 Deoxynucleoside Monophosphate Kinase (1)	25 Transducin-Alpha
9 Deoxynucleoside Monophosphate Kinase (2)	26 Elongation Factor Tu (1)
10 Nucleoside Diphosphate Kinase	27 Elongation Factor Tu (2)
11 Guanylate Kinase	28 Elongation Factor Tu (3)
12 Adenylosuccinate Synthetase	29 Elongation Factor G
13 Thymidylate Synthase	30 Purine Nucleoside Phosphorylase
14 DMSO Reductase (1)	31 Formate Dehydrogenase
15 DMSO Reductase (2)	32 Ftsz
16 Histidine Triad Nucleotide-Binding Protein	33 Jel 103
17 Glutamine Phosphoribosylpyrophosphate Amidotransferase	34 Hypoxanthine Guanine Phosphoribosyltransferase

 
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RNAse T1	DMSO Reductase
Ras	Average

 
   
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RNAse T1	DMSO Reductase
Ras	Average

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Figure-7: The folding tree of our proteins.  We made the folding tree from the information in DALI domain database.  It’s an indicator of the evolutionary history of Guanine-binding proteins.  Usually, if two proteins share similar secondary structures, they are more likely to be cousins.

Figure-8: The binding-surface tree of our proteins.  This particular tree is constructed from the density difference of aliphatic carbon between every pair of proteins.  We use it to model the guanine-binding surface relationship among them.

-----1.6.1.1.                               |       (human and rat adp-ribosylation factor)                               -----1.6.1.2.                               |       (gi-alpha-1; transducin)       -----1.6.1.-----1.6.1.3.      |                        |       (ras)      |                         -----1.6.1.4.   1.6 ---------------|       (elongation factor g)      |                         -----1.6.1.5.      |                                 (elongation factor tu)       -----1.6.3.----------1.6.3.1.                                       (adenylosuccinate synthetase)   1.11---1.11.1---------1.11.1.2.                                      (hypoxanthine-guanine-xanthine                                      phosphoribosyltransferase)                           --------1.17.1.3   1.17---1.17.1.-|           (guanylate kinase)                           --------1.17.1.4                                       (deoxynucleoside monophosphate kinase)   1.20---1.20.2.---------1.20.2.1                                       (purine nucleoside phosphorylase)               2.1.1.----------2.1.1.8.                                       (jel 103)                 5.1.7.----------5.1.7.1.                                       (nucleoside diphosphate kinase)                 48.2.1.--------48.2.1.1.                                       (formate dehydrogenase h)                   -101.1.1-----101.1.1.1.  101.1.--|                    (rnase sa)               -101.1.2-----101.1.2.1.                                    (rnase t1, rnase f1; rnase sa; rnase t1)                                     117.1.1.1.                                    (histidine triad nucleotide-binding)                                   125.2.1.1                                   (mrna capping enzyme;                                    RNA guanylyltransferase)                                    131.1.1.2.                                    (rnase1)                                       263.1.1.1.                                   (thymidylate synthase)

+-------------------------------------------------Ftsz          +-23          !  +DMSO reductase       +-28       !  !     +thimidylate synthetase       !  !  +-20       !  +-26  +--------------------------------------------------Jel 103         !    +-31     +glutamine phosphoribosylpyrophosphate amidotransferase    !  !    !  !     +DMSO reductase    !  !  +-24    !  !  !  +---------------------------------------------HINT    !  +-27    !     !  +formate dehydrogenase h    !     +-17    !        +-------------------------------------purine nucleoside    !                                                                  phosphorylase  -32guanylate kinase    !                            +-----------------------------rnase t1    !                         +--3    !                     +---5  +-----------------------------rnase ms    !                     !   !    !                 +--12   +-----------------------------------rnase f1    !                 !   !    !                 !   !  +----------------------------------DNA polymerase 1    !              +-16   +-11    !              !  !      +--------------------------------------rnase sa    !              !  !    !              !  !     +------------------------------------DNA polymerase 2    !              !  +----10    !              !        +--------------------------------Hgprtase    !              !    !              !                 +---------------- human adp-ribosylation    !              !              +--6                       factor    !              !              !  +-----------------rat adp-ribosylation    !              !           +--8                           factor    !           +-18           !  !    +-------------------ras    !           !  !           !  +----4    !           !  !        +--9       +----------------------rap    !           !  !        !  !    !           !  !        !  !       +-----------------transducin    !           !  !        !  !  +----2    !           !  !     +-13  +--7    +----------------gi-alpha-1    !           !  !     !  !     !    !           !  !     !  !     +---------------------elongation factor Tu 3    !        +-19  !  +-14  !    !        !  !  !  !  !  +------------------------------elongation factor g    !        !  !  !  !  !    !        !  !  +-15  +--------------------------------elongation factor Tu 2    !        !  !     !    !     +-22  !     !           +-----------------------deoxynucleoside    !     !  !  !     +---------1                       monophosphate kinase 1    !     !  !  !                     +-----------------------deoxynucleoside    !     !  !  !                                               monophosphate kinase 2    !  +-29  !  +adenylosuccinate synthetase    !  !  !  +----------------------------------------------rnase A    +-30  !  +mrna capping enzyme       !  +-25       !     +-------------------------------------------------------elongation       !                                                                              factor Tu 1       !  +mrna capping enzyme       +-21          +-------------------------------------------------nucleoside                                                                    diphosphate  kinase

 
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