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Activated Conformations of the ras-gene-Encoded p21 Protein. 1. An Energy-Refined Structure for the Normal p21 Protein Complexed with GDP Daryll C. Dykes
a b
c
, Paul Brandt-Rauf , Sharon M.
b
d
e
Luster , Denise Chung , Fred K. Friedman & Matthew R. Pincus
a
a
Department of Pathology and Department of Biochemistry & Molecular Biology SUNY Health Science Center at Syracuse , 750 E. Adams Street, Syracuse , NY , 13210 b
Department of Anatomy and Cell Biology SUNY Health Science Center at Syracuse , 750 E. Adams Street, Syracuse , NY , 13210 c
Division of Environmental Sciences Columbia College of Physicians & Surgeons , New York , NY , 10016 d
Department of Chemistry , New York University , New York , NY , 10003 e
Laboratory of Molecular Carcinogenesis , National Cancer Institute , Bethesda , MD , 20897 Published online: 21 May 2012.
To cite this article: Daryll C. Dykes , Paul Brandt-Rauf , Sharon M. Luster , Denise Chung , Fred K. Friedman & Matthew R. Pincus (1992) Activated Conformations of the ras-gene-Encoded p21 Protein. 1. An Energy-Refined Structure for the Normal p21 Protein Complexed with GDP, Journal of Biomolecular Structure and Dynamics, 9:6, 1025-1044, DOI: 10.1080/07391102.1992.10507977 To link to this article: http://dx.doi.org/10.1080/07391102.1992.10507977
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Journal of Biomolecular Structure & Dynamics, /SSN 0739-1102 Volume 9, Issue Number 6 (1992), If>Adenine Press (1992).
Activated Conformations of the ras-gene-Encoded p21 Protein. 1. An Energy-Refined Structure for the Normal p21 Protein Complexed with GDP Daryll C. Dykes 1'2, Paul Brandt-Raur', Sharon M. Luster\* Denise Chung\ Fred K. Friedman5 and Matthew R. Pincus ' Downloaded by [Rutgers University] at 13:23 06 April 2015
1
Department of Pathology, and Department of Biochemistry & Molecular Biology SUNY Health Science Center at Syracuse 750 E. Adams Street, Syracuse, NY, 13210 2
Department of Anatomy and Cell Biology SUNY Health Science Center at Syracuse 750 E. Adams Street, Syracuse, NY, 13210 3
Division of Environmental Sciences Columbia College of Physicians & Surgeons New York, NY, 10016 4
Department of Chemistry New York University New York, NY, 10003
5
Laboratory of Molecular Carcinogenesis National Cancer Institute Bethesda, MD, 20897
Abstract A complete three-dimensional structure for the ras-gene-encoded p21 protein with Gly 12 and Gin 61, bound to GDP, has been constructed in four stages using the available a-carbon coordinates as deposited in the Brookhaven National Laboratories Protein Data Bank. No all-atom structure has been made available despite the fact that the first crystallographic structure for the p21 protein was reported almost four years ago. In the p21 protein, if amino acid substitutions are made at any one of a number of different positions in the amino acid sequence, the protein becomes permanently activated and causes malignant transformation of normal cells or, in some cell lines, differentiation and maturation. For example, all amino acids except Gly and Pro at position 12 result in an oncogenic protein; all amino acids except Gln, Glu and Pro at position 6llikewise cause malignant transformation of cells. We have constructed our all-atom structure of the non-oncogenic protein from the x-ray structure in order to determine how oncogenic amino acid substitutions affect the three-dimensional *Author to whom correspondence should be addressed.
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structure of this protein. In Stage 1 we generated a poly-alanine backbone (except at Gly and Pro residues) through the a-carbon structure, requiring the individual Ala, Pro or Gly residues to conform to standard amino acid geometry and to form trans-planar peptide bonds. Since no a-carbon coordinates for residues 60-65 have been determined these residues were modeled by generating them in the extended conformation and then subjecting them to molecular dynamics using the computer application DISCOVER and energy minimization using DISCOVER and the ECEPP (Empirical Conformational Energies for Peptides Program). In Stage 2, the positions of residues that are homologous to corresponding residues of bacterial elongation factorTu (EF-Tu) to which p21 bears an overall40% sequence homology, were determined from their corresponding positions in a high-resolution structure ofEF-Tu. Non-homologous loops were taken from the structure generated in Stage 1 and were placed between the appropriate homologous segments so as to connect them. In Stage 3, all bad contacts that occurred in this resulting structure were removed, and the coordinates of the acarbon atoms were forced to superimpose as closely as possible on the corresponding atoms of the reference (x-ray) structure. Then the side chain positions of residues of the nonhomologous loop regions were modeled using a combination of molecular dynamics and energy minimization using DISCOVER and ECEPP respectively. All of the residues of the structure were then allowed to move under restrained energy minimization where the restraints were gradually removed. In Stage 4, the nucleotide GOP was added to the model and further energy minimization was carried out. The energy of the protein-GOP complex was minimized by allowing the atoms of GOP to move with the protein held fixed and then by allowing both the nucleotide and the residues of the protein to move together. The reconstructed model agrees with the published features of the p21 protein-GOP complex including the hydrogen bonding scheme, the distribution of backbone dihedral angles, the residues contacting the nucleotide, and the orientation ofloops with respect to one another in the protein. The structure also agrees with one that was predicted previously (Chen, J.M. et al., J. Biomol. Struct. Dynamics 6, 850-875 (1989)). In our molecular dynamics-energy minimization procedures, we also have been able to place all residues except Ala 66, which occurs in a poorlydefined region crystallographically, in local single residue minima, including residues reported to be in high energy regions in the x-ray structure. The constructed model can explain observed physical phenomena such as autophosphorylation by GTP on Thr 59 in proteins containing Thr in place of Ala 59.
Introduction The ras-oncogene-encoded p21 protein has been implicated in causing and/or promoting a number of different human tumors (1-3). The oncogenic proteins are mutated forms of proto-oncogene-encoded p21 proteins that are present in all normal eukaryotic cells. These mutated proteins differ from the normal proteins in that they contain substitutions at critical positions in the amino acid sequence (1). For example, any amino acid, except proline, that substitutes for glycine at position 12, results in an oncogenic protein (1,4,5). Other positions at which amino acid substitutions result in transforming p21 proteins include 13, 59, 61, and 63 (1 ). Positions 12 and 13 occur in a consensus sequence, (10)-Gly-X-X-X-X-Gly-Lys-(16), that comprises part of a phosphate binding site in purine nucleotide-binding proteins (1,6). Amino acids 55-70 comprise another phosphate-binding domain that is conserved in different purine nucleotide-binding proteins, including p21, bacterial elongation factor Th (EF-Tu), and adenylate kinase, (ADK) (6).
ras-p21 proteins are G-proteins which, when activated by an extracellular signal
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ras·gene Encoded p21 Protein Table I
001
--------MTEYKLVVVGAGGVGKSALTIQL--IQSYFVDE-YDPTIE-0 I II I I I I I I I I I I I I I I I I I I I I
001
SKEKFGRTKPHVNVGTIGHVDHGKTTLTAAITTVLAKTYGGAARAFDOTD 111111111
[EF-Tu
1111111
001
--------MTEYKLVVVGAGGVGKSALTIQLIQN----------- ----
[STR p21]
039
SYRKQVVIDG------ETCLLD----------------ILDTAGQEE---
[SEQ p21]
Ill
051
111111
111111
111111111
DAP-EEKARG------ITINTSHVEYDTP----TRHYAHVDCPGHAD--11111111
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[SEQ p21]
IIIIIIII IIIIII I
11111-11
[EF-Tu
027
----------HFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSA
[STR p21]
064
-YSA-MRDQYMRTGEGFLCVF-AINNTKSFED-1-HQYREQIKRVKDSDD
[SEQ p21]
Ill
087
1111
11111111111 II
II I 11111111111111
-YVKNMITG-AAQMDGAILVVAAT------DGPM-POTREHILLGROVG111111111111
II
[EF-Tu
111111111111
067
MRDQYMR-----TGEGFLCVFAIN------NTKSFEDIHQYREQIKRV--
(STR p21]
109
V----PMVLVG-NKCDL-AAR----------TYESRQAQ-----DLARSY
(SEQ p21J
I
127
111111 IIIII
II++++
11111111
111111
V----PYIIVFLNKCDMVDDQVDDEELLELVEMEARELL-(S)-QYDFPG 1111111-
1111
[EF-Tu
11111111
104
KDSDDVPMVLVGNKCDLAGR----TVESRQ----AQDLA--R--SY--G-
[STR p21]
138
G-IPYIETSA-KTRQGV-E-DA
[SEQ p21]
I 11111111 111111 I II
165
DDTPIVRGSALKALEGDAEWEAKILELAGFLDSYIP 111111-1
139
(EF-Tu
1111111111111
--IPYIETSAKT-R-----Q--GVEDAFYTLVREIRQH
[STR p21]
Identification of homologous segments by sequence between p21 and EF-Tu (lines SEQ p21 and EF-Tu) and by structure (lines STR p21 and EF-Tu). All vertical lines represent homologous residues. Structurally conserved residues are represented by regions of vertical lines between lines STR p21 and EF-Tu, while segments not connected by vertical lines represent intervening loops. The four darkened boxes represent nucleotide binding domains which are highly conserved in purine nucleotide-binding proteins.+ = residues included in the EF-Tu sequence reported in by Halliday (36) not found in the x-ray crystal structure. (S) = serine residue deleted from Halliday's EF-Tu sequence.
from a growth factor, exchange GTP forGDP. This event, in turn, is thought to result in a change in the three-dimensional structure of the protein which enables it to interact with other proteins involved in the transduction of the growth/initiator signal (1 ). Little is known about the signal transduction pathway. It has recently been shown that the region around 32-45 in p21 interacts with a GTPase activating protein (GAP) (7). In addition, p21 is known to interact with guanine nucleotide exchange factors (GNEF) (8,9). Intracellular labelling experiments indicate that overexpressed p21 protein binds to a 60 kD protein identified as a heat-sensitive protein (10,11) that may also be a GNEF. We have shown that an oncogenic p21 protein interacts with at least three different
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Legends for Color Folios
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Figure 1: Progression of construction of the three-dimensional model of normal p21 complexed with GDP; A)(Top, Left Panel) The a-carbon trace (blue) and heavy atoms ofGDP (yellow) taken directly from the PDB file for the x-ray structure; B) (Second from Top, Left Panel) a-carbon trace of regions of p21 that have structural homology to EF-Tu (purple) superimposed on the a-carbon trace of the x-ray structure of p21 (blue). The heavy atoms of the nucleotide from the x-ray structure (yellow) are included for reference. The structures of these regions correspond to Stage 2 ofthe model construction (before structural refinement); C) (Third from Top, Left Panel) Result of Stage 3 (gross and medium refinement) of the model construction. The a-carbon trace of the structure (purple) is superimposed on the x-ray structure (blue). The nucleotide (yellow) is included for reference; D) (Bottom, Left Panel) Final energy refined model for the all-atom 171-residue normal p21 protein (purple) superimposed on the a-carbon trace of the x-ray structure (blue). The final computed conformation and position ofGDP is shown in red while the conformation and position ofGDP from the x-ray structure is shown in yellow. Figure 3: (Top, Right Panel) Plot of distribution of the backbone dihedral angles , 'If) values for all of the residues in the protein over the (, 'If) map with published values for this protein. A. Comparison of the Hydrogen Bonding Schemes
A backbone-backbone hydrogen-bonding scheme between the ~-pleated sheets of the x-raycrystal structure of the Gly 12-p21 protein (residues 1-171) complexed with GDP has been published (16). Figure 2 summarizes this scheme for the computed structure and compares it with that of the published structure. As can be seen in this figure, there is generally good agreement in the hydrogen bonding schemes. B. Distribution of Polar and Non-Polar Residues
In the x-ray crystal structure of p21 complexed with GDP (16), it was reported that
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ras·gene Encoded p21 Protein
Figure 2: Comparison ofbackbone-backbone hydrogen-bonding scheme for modeled and x-ray crystal structures. Above dotted lines: comparison ofheavy atom distances between indicated atoms (model/xray structure). Below dotted lines: N-0-C angles between indicated residues (model/x-ray structure).
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most of the side chains of the non-polar residues were inaccessible to solvent except for Ala 121, Ala 122, and Leu 171. We find that all of the non-polar residues are buried except for Leu 120, Ala 122, Val125, Leu 168 and Leu 171. The latter two residues occur at the carboxyl-terminal end of the truncated protein and may be not be exposed in the complete (189-residue) form. The minor discrepancies regarding Leu 120,Ala 121, and Va1125 may relate to different rotational isomeric states for the side chains of these residues. It is clear from our model that Leu 120,Ala 122, and Val 125, all of which occurin bend regions, may be exposed and may contribute to a contact site for interaction with other intracellular proteins.
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C. Backbone Dihedral Angle Distribution
Figure 3 summarizes a comparison of the distribution of the backbone dihedral angles, and"'' between our computed structure and the X-ray crystal structure. As can be seen from this comparison, there is general agreement between the two frequency distributions. However, some differences occur. Three residues occur in high energy regions in the x-ray structure (16), viz., Ser 65 (H), Ala 122 (H) and Arg 123 (H). (See ref. 34 for definitions of backbone states.) In our energy-refined structure, all of these residues occur in low energy regions: Ser 65 (A*), Ala 122 (D) and Arg 123 (C). In fact, only one residue in our computed structure occurred in a high energy region, Ala 66 (H). This residue occurs in a poorly-defined region of the protein (14-17). Thus, the energy-refined structure results in placing residues in low energy single residue minima (34,35). In a recent survey of high resolution x-ray crystal structures, it was found that only few amino acid residues adopted energetically unfavorable conformations on the {Q>, 'I') map (40). Our results are compatible with these findings. Structures of Non-Homologous Regions
There are nine regions of the p21 protein that have no sequence or structural homology to any regions ofEF-Tu (see Table I). These regions were modeled by using a systematic procedure in which they were subjected to high temperature dynamics, followed by a step-wise temperature-lowering process, followed by energy minimization. All of these procedures contained the requirements that the generated acarbon coordinates superimpose on those of the x-ray structure, thatthe geometry of individual residues conform to the standard geometry for these residues, that peptide bonds be trans-planar and that the backbone conformation occur in a region of low energy for the single residue minima (34). A representation ofthe loop structures is shown in Figure 4. It is clear that these loop regions occur near the surface of the protein. These segments are potential sites for contacting other intracellular proteins in the signal transduction pathway induced by activated p21. It should be noted that residues 102-103 have recently been proposed as being
involved in nucleotide exchange (41 ). These residues occur on the surface of the protein but occur in a structurally conserved region corresponding to residues 124-125
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ras·gene Encoded p21 Protein HN (M).Val14 HN(M)-Giy 15 3.35
.J
HN-Lys 16
3 09
HO
0
I
I
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~~ 13 rJ,:- il'·" HN(M)-Giy15
_ 2 94 HO-Ser 17
2.91
?-Asp 30
J::5-
3.31
3.30
0
I
0
HN(M)-Aia 18
I
3.64
OH
,_,.f
N~
NH~o,
~H'-~9
HN-Lys 117
3.08 HN(M)-Ser 17
H
,2.95
0 ,C-.... 1I9
~HN(M)-Aia 146
HN-Asn 116
Figure 5: Interactions between p2l and GDP. Lines connecting atoms of the protein and atoms of the nucleotide represent possible hydrogen bonding interactions. The distance (in angstroms) between interacting atoms are indicated next to each line.
in EF-Tu. Possibly these residues in EF-Tu are likewise involved in interacting with specific nucleotide exchange factors.
Structure of the Nucleotide Binding Domains The residues involved in binding GTP and G DP may be divided into those interacting with the guanine base, those interacting with the ribofuranosyl ring and those interacting with the phosphates. As shown in Figure 5, the residues involved in binding the base and ribose rings are the same as those reported in the x-ray crystal structure (16). Of major interest is the orientation and conformation of the two major domains involved in binding to the ~-phosphate moiety involving residues 6-17 and 55-70. It was predicted previously that in the lowest energy structure for the normal or inactive form of the Gly 12-containing protein, a bend can occur at Ala 11-Gly 12 (5). A higher energy structure containing a bend at Gly 12-Gly 13 was predicted to occur both in the activated normal and the transforming proteins (5). In the stereo drawing of the normal p21 protein bound toG DP presented in ref. 17, there appears to be a bend at Ala 11-Gly 12. In the structure for the same protein from the PDB used in this study, the bend is found to occur at Gly 12-Gly 13. Possibly this dichotomy of results may be due to the existence of these two predicted forms. In permanently activated oncogenic proteins containing substitutions at position 12, the bend was predicted to occur at positions 12 and 13 only (5). In the x-ray crystal structure ofVal 12-p21 protein bound to GDP (16), the bend occurs at Val 12-Gly 13 as predicted. The current x-ray crystallographic results tend, therefore, to confirm the earlier predictions regarding these residues (5). In a previous publication (6), we predicted that the positioning of two phosphatebinding regions, viz. 6-17 and 55-70, with respect to one another, would be identical
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to the relationship of the corresponding regions ofEF-Tu (residues 14-25 and 78-93, respectively) and ADK (residues 11-22 and 92-107, respectively). We now find in our constructed p21 model that both the relative positions and conformations of the segments are quite similar to one another as we reported for our previously constructed model (see ref 6 for a comparison of the structures). The region of p21 from residues 61-70 has the highest temperature factor in the protein (14-17), and the region from residues 59-69 in the x-ray crystallographic structure (16) was not well-defined due to its high mobility. In the deposited coordinates for p21 in the PDB, no a-carbon coordinates for residues 61-65 were determined due to the low resolution of the residues in this region although these residues are included in the published a-carbon trace (16). In previous calculations of the lowest energy structures for the p21 segment of residues 55-67 (28), we found that residues 62-67 formed an a-helix and that residues 55-58 were extended. A bend was found to occur at residues 59-60 and G1n 61 adopted the C7 equatorial conformation (28). This conformation was found to be identical to the corresponding regions ofEF-Tu and ADK (6). In the reconstructed structure of the p21 protein, the sequence from 55-58 is extended while the segment from 62-67 is helical, in agreement with the previous prediction (6).
Orientation ofthe Side Chain of Gin 61 In computing the structure of the Gin 61 region, use was made of the homology between residues 52-63 of p21 and 75-86 of EF-Tu. In computing the low-energy positions for the side chains of Gin 61, for an initial structure, it was assumed that the side chain atoms ofGln would be superimposable on the corresponding atoms ofHis 84. Energy minimization of the position of this side chain resulted in a stable conformation in which this side chain points away from the ~-phosphate ofGDP. In thex-raystructureofp21 complexed with GTP,itwas reported thatthis side chain actually extends into the phosphate-binding pocket (14,15). Dynamic simulations carried out on the computed lowest-energy conformation of the Gln 61 side chain resulted in several low-energy conformations in which the side chain points into the phosphate-binding pocket. It has been surmised that the -NH2 group of this residue may be important in the catalytic mechanism possibly by interacting with a nucleophilic water molecule in the active site (14,15). This mechanism cannot be a general one, however, because Pro and Glu can both substitute for Gin 61 without resulting in an oncogenic protein (43). Neither of these latter two residues contains a side chainNH 2 group. It is therefore unclear as to the exact role of this residue and to the exact positioning of its side chain. These considerations indicate that there are likely many low energy conformations for the side chains of amino acids in proteins - especially for polar residues on the surface. The above results also suggest that modeling of protein structure on the basis of homology can give different side chain positions from those seen in the xray crystal structure, and it is likely that both the computed and x-ray conformations are available to the side chain of Gin 61.
ras-gene Encoded p21 Protein
1041
Effects of Substitutions at Positions 59 It was shown in a preceding publication (27), that substitution ofThr for Ala 59 in the predicted structure would place the y-OH group of the Thr residue so that it would point into the phosphate binding pocket and would be aligned with the yphosphate ofGTP bound to the protein. Viral forms of the p21 protein contain this substitution and are known to undergo autophosphorylation by GTP (1). This alignment of the Thr residue is preserved in the current model, explaining how this residue can undergo phosphorylation by GTP in the autophosphorylation process.
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Conformation of the Effector Loop Region Residues 32-40 ofp21 have been implicated in interacting with GAP when the protein binds GTP (7). As the result of previous calculations on the structure of activated p21 proteins (27), the region from residues 35-47 was found to move significantly. The p21 peptide from 35-47 was synthesized recently and found to block oncogenic Val 12-p21 protein-induced maturation of oocytes (30). Control peptides of similar chain length were found not to inhibit the ras-effect, however. The structure of the peptide from 35-47 is shown in Figure 6. It may be noted that the conformation is an extended one. There appears to be a segregation of charges into two negative charges (Glu 37 and Asp 38; shown in red) and two positive charges (Arg 41 and Lys 42; shown in blue). The charged region is followed by a discrete hydrophobic region consisting of Val 44, Val 45, and Ile 46 (shown in green). The segregated charges and hydrophobic regions in the extended conformation may be important in the binding of p21 to intracellular effector proteins.
Limitations of the Method While the methods used in construction of the whole protein from the a-carbon trace result in the construction of a reasonable structure, there are limitations to this methodology. First, the effects of solvation have been omitted from these calculations. Solvation has been shown to be critical in the folding of the p21 protein and even for isolated segments of this protein (28). However, the modeling process was subjected to two general sets of requirements that allow exclusion of the effects of solvation: the a-carbon coordinates were required to remain close to those of the x-ray structure (except for the isolated short segment, residues 60-65 for which no a-carbon coordinates were available), and residues homologous to those in EF-Tu, a protein of very similar structure to p21 (20), were required to adopt similar conformations. Previous calculations based purely on sequence homology without even the first set of requirements resulted in successful prediction of the structure of a protein from its homologue of known structure (23) without consideration of the effects of hydration. Second, construction of the structure of p21 was based largely on its homology with EF-Tu. While there is overall forty percent sequence homology and while there is considerable structural homology between the two proteins, there are significant differences between the two proteins. For example, in structurally conserved regions,
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there are significant differences in sequence. In the phosphate-binding loop consensus sequence, the corresponding sequences for both proteins are: Gly-Ala-Gly-Gly-Val-Gly-Lys (10-16, p21)
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Gly-His-Val-Asp-His-Gly-Lys (18-24, EF-Tu) Moreover, as noted in the previous section, modelling of the side chain ofGln 61 on the basis of the conformation of its structural homologue, His 84, in EF-Tu, resulted in its pointing away from the phosphate binding site. Modeling of the side chains of p21 from the corresponding side chains ofEF-Tu in these regions is less valid than in regions where the sequence homology is greater. In these regions, therefore, the restraints holding the positions of the side chains to those of the corresponding side chains ofEF-Tu were reduced in the molecular dynamics and energy minimization procedures. In addition, the constraints involving the a-carbon coordinates tend to limit the number of possible side chain conformations for given backbone conformations. Also for the side chain of Gin 61, it is probable that this side chain can adopt a number oflow energy conformations, and it is apparently not necessary for its side chain to point into the active site as discussed in the preceding section. Third, the use of two sets of potentials, from DISCOVER and ECEPP, may conceivably lead to incompatibilities in results. In a recent comparison of potential functions used to compute peptide and protein structure (40), it was shown that three different sets of potential functions could result in major differences in the results of computing peptide structure. ECEPP was found to reproduce the single residue minima and also to predict the root-mean square end-to-end distance for poly-Ala most accurately (40). While DISCOVER was not included in this test of potentials, this program allows for bond angle bending and bond stretching which was shown to distort the (, 'I') map using the other potential functions (40). Because of this distortion, the C-7 axial conformation was found to be a low energy one with the potential functions tested (other than ECEPP) despite the fact that it is only rarely observed in protein structures (40) and is predicted to be energetically forbidden by ECEPP which does not allow distortion of bond angles and bond lengths. However, in our calculations, individual residues were required to adopt low energy conformations as defined by ECEPP. This process resulted in placement of residues like Arg 123, formerly in a high energy region, into one of the low energy single residue minima. The main use of DISCOVER was to generate representative samplings oflow energy minima for peptide segments (or side chains) that were already subjected to the constraints described above and in the Methods section. Fourth, there may be inaccuracies in the coordinates from the x-ray structure especially in the 60-65 region. The entire region from residue 59-69 is not welldefined due to high thermal fluctuations (14-17). Thus the given a-carbon coordinates in the entire region may not be as accurate as for other regions of the protein. However, the corresponding region ofEF-Tu is better defined crystallographically (25) so that homology of the residues ofp21 in this region to those ofEF-Tu allows for construction of more accurate coordinates for these residues. Nonetheless,
ras·gene Encoded p21 Protein
1043
further refinement of this region is being undertaken and will include the effect of solvation to determine a more accurate structure. Our coordinates for all of the atoms of residues 1-171 ofp21 complexed with GOP have been submitted to the Brookhaven National Laboratory Protein Data Base.
Acknowledgement This work was supported in part by an American Heart Association Fellowship to DCD, and NIH Grant CA42500 to MRP.
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References and Footnotes
1. Barbacid, M.,Ann. Rev. Biochem. 56,779-827 (1987). 2. Almoguerra, C., Shibata, D., Forrester, K., Martin, J., Arnheim, M. and Perucho, M., Cell 53, 549554 (1988). 3. Forrester, K., Almoguerra, C., Han, K., Grizzle, W., and Perucho, M., Nature 327, 298-303 (1987). 4. Seeburg, P.H., Colby, W.W., Capon, D.J., Goedde!, D.V. and Levinson, AD., Nature (London) 312, 71-75 (1984). 5. Pincus, M.R., van Renswoode, J., Harford,J.B., Chang, E.H., Carty, R.P. and Klausner, R.D.,Proc. Nat/. Acad. Sci. USA 80, 5253-5257 (1983). 6. Chen, J.M., Lee, G., Brandt-Rauf, P.W., Murphy, R.B., Gibson, K.D., Scheraga, HA, Rackovsky, S. and Pincus, M.R., Int. J, Peptide Protein Res. 36, 247-254 ( 1990). 7. Adari, H., Lowy, D.R., Willumsen, B.M., Der, C.J., and McCormick, F., Science 240, 518-520 (1988). 8. West, M., Kung, H.-F., and Kamata, T., Febs. Lett. 259,245-248 (1990). 9. Woffman, A and Macara, I.G., Science 248,67-69 (1990). 10. deGunzburg, J., Riehl, R. and Weinburg, R.A, Proc. Nat/. Acad. Sci. USA 86, 4007-4011 (1989). 11. Downward, J., Riehl, R., Wu, L. and Weinberg, R.A, Proc. Nat/. Acad. Sci. USA 87, 5998-6002 (1990). 12. Lee, G., Ronai, ZA, Pincus, M.R., Brandt-Rauf, P.W., Murphy, R.B., Delohery, T.M., Nishimura, S., Yamaizumi, Z. and Weinstein, I.B., Proc. Nat/. Acad. Sci. USA 86, 8678-8682 (1989). 13. Chung, D.L., Brandt-Rauf, P.W., Weinstein, I.B., Nishimura, S., Yamaizumi, Z., Murphy, R.B., and Pincus, M.R., Proc. Nat/. Acad. Sci., in press (1992). 14. Schlichting, I.,Almo, S.C., Rapp, G., Wilson, K., Petratos, K., Lentfer,A, Wittinghofer, A, Kabsch, W., Pai, E.F., Petsko, GA and Goody, R.S., Nature (London) 345, 309-315 (1990). 15. Krengel, N., Schlichting, I. Scherer, A, Schumann, r. Frech, M., John,J., Kabsch, W., Pai, E.F., and Wittinghofer, A, Cell62, 539-548 (1990). 16. Tong, L., de Vos, AM., Milburn, M.V., and Kim, S.-H.,J Mol. Bioi. 217,503-516 (1991). 17. Brunger, AT., Milburn, M.V., Tong, L., de Vos, A, Jancarik, J., Yamaizumi, Z., Nishimura, S., Ohtsuka, E. and Kim, S.H., Proc. Nat/. Acad. Sci. USA 87,4849-4853 (1990). 18. Willumsen, B.M. Papageorge, A. G., Kung, H.-F., Beckesi, E., Robins, T., Johnsen, M., Vass, W.C. and Lowy, D.R., Mol. Cell. Bioi. 6, 2646-2654 ( 1986). 19. Casey, PJ., Solski, PA, Der, CJ., and Buss, J.E., Proc. Nat/. Acad Sci. USA 86,8323-8327 (1989). · 20. Holbrook, S.R. and Kim, S.-H., Proc. Nat/. Acad. Sci. USA 86, 1751-1753 (1989). 21. Tong, L., de Vos,AM., Milburn, M.V., Jancarik, J., Noguchi, S., Nishimura, S., Miura, K., Ohtsuka, E. and Kim, S.-H., Nature (London) 337,90-93 (1989). 22. de Vos,AM., Tong, L., Milburn, M.V. Matias, P.M.,Jancarik,J., Noguchi, S., Nishimura, S., Miura, K., Ohtsuka, E., and Kim, S.-H., Science 239, 888-893 (1988). 23. Warme, P.K., Momany, FA, Rumball, S.V., Tuttle, R.W. and Scheraga 13, 768-782 (1974). 24. Smith, S.G., Lewis, M., Aschaffenburg, R., Fenna, R.E., Wilson, lA Sundaralingham, M., Stuart, D.l. and Phillips, D.C., Biochem. J 242, 353-360 (1987). 25. LaCour, T.F.M., Nyborg, J., Thirup, S., and Clark, B. F. C., EMBO J 4, 2385-2388 (1985). 26. Hagler, AT., Osguthorpe, DJ., Dauber-Osguthorpe, P., Hemple, J.C., Science 227, 1309-1315 (1985).
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27. Chen, J.M., Lee, G., Murphy, R.B., Carty, R.P., Brandt-Rauf, P.W., Friedman, E., and Pincus, M.R.,J Biomol. Struct. Dynamics 6, 859-875 (1988). 28. Pincus, M.R., Brandt-Rauf, P.W., Gibson, K, Carty, R.P., Lubowsky, J., Avitable, M. and Scheraga, H.A, Proc. Nat/. Acad. Sci. USA 89, 8375-8379 (1987). 29. Vogel, V.S., Dixon, R.AF., Schaber, M.D., Diehl, R.E., Marshall, M.S., Scolnick, E.M., Sigal, I.S., and Gibbs, J.B., Nature (London) 335, 90-93 (1988). 30. Chung, D.L., Brandt-Rauf, P.W., Murphy, R.B., Nishimura, S., Yamaizumi, Z., Weinstein, I.B., and Pincus, M.R.,Anticancer Res. 11, 1373-1378 (1991). 31. Clanton, D.J., Lu, Y., Blair, D.G., and Shih, T.Y., Mol. Cell. Bioi. 7, 3092-3097 (1987). 32. Neal, S.E., Eccleston, J.F., and Webb, M.R., Proc. Nat/. Acad. Sci. USA 87,3562-3565 (1990). 33. Nemethy, G., Pottle, M.S. and Scheraga, H.A,.J. Phys. Chern. 87, 1883-1887 (1983). 34. Zimmerman, S.S., Pottle, M.S., Nemethy, G., and Scheraga, H.A,Macromolecules 10, 1-9 (1977). 35. Vasquez, M., Nemethy, G., and Scheraga, H.A, Macromolecules 16, 1043-1049 (1983). 36. Halliday, KR., J. Cyclic Nucleotide and Protein Phosphorylation Research 9, 435-448 (1983). 37. Valencia, A, Kjelgaard, M., Pai, E.F., and Sander, C., Proc. Natl. Acad. Sci. USA 88, 5443-5447 (1991). 38. Glasser, L. and Scheraga, H.A, J. Mol. Bioi. 199, 513-524 (1988). 39. Brunger,AT.,Clore,G.M.,Gronenborn,AM., and Karplus,M.,Proc.Natl.Acad. Sci. USA 83,38013805 (1986). 40. Roterman, I.K., Lambert, M. H., Gibson, K.D., and Scheraga, H.A,J. Biomol. Struct. Dynamics 7, 421-453 (1989). 41. Willumsen, B.M., Vass, W.C., Velu, T.J., Papageorge, AG., Schiller, J.T., and Lowy, D.R.,Moi Cell Bioi. 11, 6026-6033 (1991 ). 42. Jurnak, F., Science 230, 32-36 (1985). 43. Der, C.J., Finkel, T., and Cooper, G.M., Ce1144, 167-176 (1986). 44. Chen, J.M., Lee, G., Brandt-Rauf, P.W., Murphy, R.B., Rackovsky, S. and Pincus, M.R.,J. Protein Chern. 9, 543-547 (1990).
Date Received: January 20, 1992
Communicated by the Editor R.H. Sarma
Journal of Biomolecular Structure and Dynamics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsd20
Activated Conformations of the ras-gene-Encoded p21 Protein. 1. An Energy-Refined Structure for the Normal p21 Protein Complexed with GDP Daryll C. Dykes
a b
c
, Paul Brandt-Rauf , Sharon M.
b
d
e
Luster , Denise Chung , Fred K. Friedman & Matthew R. Pincus
a
a
Department of Pathology and Department of Biochemistry & Molecular Biology SUNY Health Science Center at Syracuse , 750 E. Adams Street, Syracuse , NY , 13210 b
Department of Anatomy and Cell Biology SUNY Health Science Center at Syracuse , 750 E. Adams Street, Syracuse , NY , 13210 c
Division of Environmental Sciences Columbia College of Physicians & Surgeons , New York , NY , 10016 d
Department of Chemistry , New York University , New York , NY , 10003 e
Laboratory of Molecular Carcinogenesis , National Cancer Institute , Bethesda , MD , 20897 Published online: 21 May 2012.
To cite this article: Daryll C. Dykes , Paul Brandt-Rauf , Sharon M. Luster , Denise Chung , Fred K. Friedman & Matthew R. Pincus (1992) Activated Conformations of the ras-gene-Encoded p21 Protein. 1. An Energy-Refined Structure for the Normal p21 Protein Complexed with GDP, Journal of Biomolecular Structure and Dynamics, 9:6, 1025-1044, DOI: 10.1080/07391102.1992.10507977 To link to this article: http://dx.doi.org/10.1080/07391102.1992.10507977
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Journal of Biomolecular Structure & Dynamics, /SSN 0739-1102 Volume 9, Issue Number 6 (1992), If>Adenine Press (1992).
Activated Conformations of the ras-gene-Encoded p21 Protein. 1. An Energy-Refined Structure for the Normal p21 Protein Complexed with GDP Daryll C. Dykes 1'2, Paul Brandt-Raur', Sharon M. Luster\* Denise Chung\ Fred K. Friedman5 and Matthew R. Pincus ' Downloaded by [Rutgers University] at 13:23 06 April 2015
1
Department of Pathology, and Department of Biochemistry & Molecular Biology SUNY Health Science Center at Syracuse 750 E. Adams Street, Syracuse, NY, 13210 2
Department of Anatomy and Cell Biology SUNY Health Science Center at Syracuse 750 E. Adams Street, Syracuse, NY, 13210 3
Division of Environmental Sciences Columbia College of Physicians & Surgeons New York, NY, 10016 4
Department of Chemistry New York University New York, NY, 10003
5
Laboratory of Molecular Carcinogenesis National Cancer Institute Bethesda, MD, 20897
Abstract A complete three-dimensional structure for the ras-gene-encoded p21 protein with Gly 12 and Gin 61, bound to GDP, has been constructed in four stages using the available a-carbon coordinates as deposited in the Brookhaven National Laboratories Protein Data Bank. No all-atom structure has been made available despite the fact that the first crystallographic structure for the p21 protein was reported almost four years ago. In the p21 protein, if amino acid substitutions are made at any one of a number of different positions in the amino acid sequence, the protein becomes permanently activated and causes malignant transformation of normal cells or, in some cell lines, differentiation and maturation. For example, all amino acids except Gly and Pro at position 12 result in an oncogenic protein; all amino acids except Gln, Glu and Pro at position 6llikewise cause malignant transformation of cells. We have constructed our all-atom structure of the non-oncogenic protein from the x-ray structure in order to determine how oncogenic amino acid substitutions affect the three-dimensional *Author to whom correspondence should be addressed.
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structure of this protein. In Stage 1 we generated a poly-alanine backbone (except at Gly and Pro residues) through the a-carbon structure, requiring the individual Ala, Pro or Gly residues to conform to standard amino acid geometry and to form trans-planar peptide bonds. Since no a-carbon coordinates for residues 60-65 have been determined these residues were modeled by generating them in the extended conformation and then subjecting them to molecular dynamics using the computer application DISCOVER and energy minimization using DISCOVER and the ECEPP (Empirical Conformational Energies for Peptides Program). In Stage 2, the positions of residues that are homologous to corresponding residues of bacterial elongation factorTu (EF-Tu) to which p21 bears an overall40% sequence homology, were determined from their corresponding positions in a high-resolution structure ofEF-Tu. Non-homologous loops were taken from the structure generated in Stage 1 and were placed between the appropriate homologous segments so as to connect them. In Stage 3, all bad contacts that occurred in this resulting structure were removed, and the coordinates of the acarbon atoms were forced to superimpose as closely as possible on the corresponding atoms of the reference (x-ray) structure. Then the side chain positions of residues of the nonhomologous loop regions were modeled using a combination of molecular dynamics and energy minimization using DISCOVER and ECEPP respectively. All of the residues of the structure were then allowed to move under restrained energy minimization where the restraints were gradually removed. In Stage 4, the nucleotide GOP was added to the model and further energy minimization was carried out. The energy of the protein-GOP complex was minimized by allowing the atoms of GOP to move with the protein held fixed and then by allowing both the nucleotide and the residues of the protein to move together. The reconstructed model agrees with the published features of the p21 protein-GOP complex including the hydrogen bonding scheme, the distribution of backbone dihedral angles, the residues contacting the nucleotide, and the orientation ofloops with respect to one another in the protein. The structure also agrees with one that was predicted previously (Chen, J.M. et al., J. Biomol. Struct. Dynamics 6, 850-875 (1989)). In our molecular dynamics-energy minimization procedures, we also have been able to place all residues except Ala 66, which occurs in a poorlydefined region crystallographically, in local single residue minima, including residues reported to be in high energy regions in the x-ray structure. The constructed model can explain observed physical phenomena such as autophosphorylation by GTP on Thr 59 in proteins containing Thr in place of Ala 59.
Introduction The ras-oncogene-encoded p21 protein has been implicated in causing and/or promoting a number of different human tumors (1-3). The oncogenic proteins are mutated forms of proto-oncogene-encoded p21 proteins that are present in all normal eukaryotic cells. These mutated proteins differ from the normal proteins in that they contain substitutions at critical positions in the amino acid sequence (1). For example, any amino acid, except proline, that substitutes for glycine at position 12, results in an oncogenic protein (1,4,5). Other positions at which amino acid substitutions result in transforming p21 proteins include 13, 59, 61, and 63 (1 ). Positions 12 and 13 occur in a consensus sequence, (10)-Gly-X-X-X-X-Gly-Lys-(16), that comprises part of a phosphate binding site in purine nucleotide-binding proteins (1,6). Amino acids 55-70 comprise another phosphate-binding domain that is conserved in different purine nucleotide-binding proteins, including p21, bacterial elongation factor Th (EF-Tu), and adenylate kinase, (ADK) (6).
ras-p21 proteins are G-proteins which, when activated by an extracellular signal
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ras·gene Encoded p21 Protein Table I
001
--------MTEYKLVVVGAGGVGKSALTIQL--IQSYFVDE-YDPTIE-0 I II I I I I I I I I I I I I I I I I I I I I
001
SKEKFGRTKPHVNVGTIGHVDHGKTTLTAAITTVLAKTYGGAARAFDOTD 111111111
[EF-Tu
1111111
001
--------MTEYKLVVVGAGGVGKSALTIQLIQN----------- ----
[STR p21]
039
SYRKQVVIDG------ETCLLD----------------ILDTAGQEE---
[SEQ p21]
Ill
051
111111
111111
111111111
DAP-EEKARG------ITINTSHVEYDTP----TRHYAHVDCPGHAD--11111111
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[SEQ p21]
IIIIIIII IIIIII I
11111-11
[EF-Tu
027
----------HFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSA
[STR p21]
064
-YSA-MRDQYMRTGEGFLCVF-AINNTKSFED-1-HQYREQIKRVKDSDD
[SEQ p21]
Ill
087
1111
11111111111 II
II I 11111111111111
-YVKNMITG-AAQMDGAILVVAAT------DGPM-POTREHILLGROVG111111111111
II
[EF-Tu
111111111111
067
MRDQYMR-----TGEGFLCVFAIN------NTKSFEDIHQYREQIKRV--
(STR p21]
109
V----PMVLVG-NKCDL-AAR----------TYESRQAQ-----DLARSY
(SEQ p21J
I
127
111111 IIIII
II++++
11111111
111111
V----PYIIVFLNKCDMVDDQVDDEELLELVEMEARELL-(S)-QYDFPG 1111111-
1111
[EF-Tu
11111111
104
KDSDDVPMVLVGNKCDLAGR----TVESRQ----AQDLA--R--SY--G-
[STR p21]
138
G-IPYIETSA-KTRQGV-E-DA
[SEQ p21]
I 11111111 111111 I II
165
DDTPIVRGSALKALEGDAEWEAKILELAGFLDSYIP 111111-1
139
(EF-Tu
1111111111111
--IPYIETSAKT-R-----Q--GVEDAFYTLVREIRQH
[STR p21]
Identification of homologous segments by sequence between p21 and EF-Tu (lines SEQ p21 and EF-Tu) and by structure (lines STR p21 and EF-Tu). All vertical lines represent homologous residues. Structurally conserved residues are represented by regions of vertical lines between lines STR p21 and EF-Tu, while segments not connected by vertical lines represent intervening loops. The four darkened boxes represent nucleotide binding domains which are highly conserved in purine nucleotide-binding proteins.+ = residues included in the EF-Tu sequence reported in by Halliday (36) not found in the x-ray crystal structure. (S) = serine residue deleted from Halliday's EF-Tu sequence.
from a growth factor, exchange GTP forGDP. This event, in turn, is thought to result in a change in the three-dimensional structure of the protein which enables it to interact with other proteins involved in the transduction of the growth/initiator signal (1 ). Little is known about the signal transduction pathway. It has recently been shown that the region around 32-45 in p21 interacts with a GTPase activating protein (GAP) (7). In addition, p21 is known to interact with guanine nucleotide exchange factors (GNEF) (8,9). Intracellular labelling experiments indicate that overexpressed p21 protein binds to a 60 kD protein identified as a heat-sensitive protein (10,11) that may also be a GNEF. We have shown that an oncogenic p21 protein interacts with at least three different
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Legends for Color Folios
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Figure 1: Progression of construction of the three-dimensional model of normal p21 complexed with GDP; A)(Top, Left Panel) The a-carbon trace (blue) and heavy atoms ofGDP (yellow) taken directly from the PDB file for the x-ray structure; B) (Second from Top, Left Panel) a-carbon trace of regions of p21 that have structural homology to EF-Tu (purple) superimposed on the a-carbon trace of the x-ray structure of p21 (blue). The heavy atoms of the nucleotide from the x-ray structure (yellow) are included for reference. The structures of these regions correspond to Stage 2 ofthe model construction (before structural refinement); C) (Third from Top, Left Panel) Result of Stage 3 (gross and medium refinement) of the model construction. The a-carbon trace of the structure (purple) is superimposed on the x-ray structure (blue). The nucleotide (yellow) is included for reference; D) (Bottom, Left Panel) Final energy refined model for the all-atom 171-residue normal p21 protein (purple) superimposed on the a-carbon trace of the x-ray structure (blue). The final computed conformation and position ofGDP is shown in red while the conformation and position ofGDP from the x-ray structure is shown in yellow. Figure 3: (Top, Right Panel) Plot of distribution of the backbone dihedral angles , 'If) values for all of the residues in the protein over the (, 'If) map with published values for this protein. A. Comparison of the Hydrogen Bonding Schemes
A backbone-backbone hydrogen-bonding scheme between the ~-pleated sheets of the x-raycrystal structure of the Gly 12-p21 protein (residues 1-171) complexed with GDP has been published (16). Figure 2 summarizes this scheme for the computed structure and compares it with that of the published structure. As can be seen in this figure, there is generally good agreement in the hydrogen bonding schemes. B. Distribution of Polar and Non-Polar Residues
In the x-ray crystal structure of p21 complexed with GDP (16), it was reported that
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ras·gene Encoded p21 Protein
Figure 2: Comparison ofbackbone-backbone hydrogen-bonding scheme for modeled and x-ray crystal structures. Above dotted lines: comparison ofheavy atom distances between indicated atoms (model/xray structure). Below dotted lines: N-0-C angles between indicated residues (model/x-ray structure).
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most of the side chains of the non-polar residues were inaccessible to solvent except for Ala 121, Ala 122, and Leu 171. We find that all of the non-polar residues are buried except for Leu 120, Ala 122, Val125, Leu 168 and Leu 171. The latter two residues occur at the carboxyl-terminal end of the truncated protein and may be not be exposed in the complete (189-residue) form. The minor discrepancies regarding Leu 120,Ala 121, and Va1125 may relate to different rotational isomeric states for the side chains of these residues. It is clear from our model that Leu 120,Ala 122, and Val 125, all of which occurin bend regions, may be exposed and may contribute to a contact site for interaction with other intracellular proteins.
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C. Backbone Dihedral Angle Distribution
Figure 3 summarizes a comparison of the distribution of the backbone dihedral angles, and"'' between our computed structure and the X-ray crystal structure. As can be seen from this comparison, there is general agreement between the two frequency distributions. However, some differences occur. Three residues occur in high energy regions in the x-ray structure (16), viz., Ser 65 (H), Ala 122 (H) and Arg 123 (H). (See ref. 34 for definitions of backbone states.) In our energy-refined structure, all of these residues occur in low energy regions: Ser 65 (A*), Ala 122 (D) and Arg 123 (C). In fact, only one residue in our computed structure occurred in a high energy region, Ala 66 (H). This residue occurs in a poorly-defined region of the protein (14-17). Thus, the energy-refined structure results in placing residues in low energy single residue minima (34,35). In a recent survey of high resolution x-ray crystal structures, it was found that only few amino acid residues adopted energetically unfavorable conformations on the {Q>, 'I') map (40). Our results are compatible with these findings. Structures of Non-Homologous Regions
There are nine regions of the p21 protein that have no sequence or structural homology to any regions ofEF-Tu (see Table I). These regions were modeled by using a systematic procedure in which they were subjected to high temperature dynamics, followed by a step-wise temperature-lowering process, followed by energy minimization. All of these procedures contained the requirements that the generated acarbon coordinates superimpose on those of the x-ray structure, thatthe geometry of individual residues conform to the standard geometry for these residues, that peptide bonds be trans-planar and that the backbone conformation occur in a region of low energy for the single residue minima (34). A representation ofthe loop structures is shown in Figure 4. It is clear that these loop regions occur near the surface of the protein. These segments are potential sites for contacting other intracellular proteins in the signal transduction pathway induced by activated p21. It should be noted that residues 102-103 have recently been proposed as being
involved in nucleotide exchange (41 ). These residues occur on the surface of the protein but occur in a structurally conserved region corresponding to residues 124-125
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ras·gene Encoded p21 Protein HN (M).Val14 HN(M)-Giy 15 3.35
.J
HN-Lys 16
3 09
HO
0
I
I
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~~ 13 rJ,:- il'·" HN(M)-Giy15
_ 2 94 HO-Ser 17
2.91
?-Asp 30
J::5-
3.31
3.30
0
I
0
HN(M)-Aia 18
I
3.64
OH
,_,.f
N~
NH~o,
~H'-~9
HN-Lys 117
3.08 HN(M)-Ser 17
H
,2.95
0 ,C-.... 1I9
~HN(M)-Aia 146
HN-Asn 116
Figure 5: Interactions between p2l and GDP. Lines connecting atoms of the protein and atoms of the nucleotide represent possible hydrogen bonding interactions. The distance (in angstroms) between interacting atoms are indicated next to each line.
in EF-Tu. Possibly these residues in EF-Tu are likewise involved in interacting with specific nucleotide exchange factors.
Structure of the Nucleotide Binding Domains The residues involved in binding GTP and G DP may be divided into those interacting with the guanine base, those interacting with the ribofuranosyl ring and those interacting with the phosphates. As shown in Figure 5, the residues involved in binding the base and ribose rings are the same as those reported in the x-ray crystal structure (16). Of major interest is the orientation and conformation of the two major domains involved in binding to the ~-phosphate moiety involving residues 6-17 and 55-70. It was predicted previously that in the lowest energy structure for the normal or inactive form of the Gly 12-containing protein, a bend can occur at Ala 11-Gly 12 (5). A higher energy structure containing a bend at Gly 12-Gly 13 was predicted to occur both in the activated normal and the transforming proteins (5). In the stereo drawing of the normal p21 protein bound toG DP presented in ref. 17, there appears to be a bend at Ala 11-Gly 12. In the structure for the same protein from the PDB used in this study, the bend is found to occur at Gly 12-Gly 13. Possibly this dichotomy of results may be due to the existence of these two predicted forms. In permanently activated oncogenic proteins containing substitutions at position 12, the bend was predicted to occur at positions 12 and 13 only (5). In the x-ray crystal structure ofVal 12-p21 protein bound to GDP (16), the bend occurs at Val 12-Gly 13 as predicted. The current x-ray crystallographic results tend, therefore, to confirm the earlier predictions regarding these residues (5). In a previous publication (6), we predicted that the positioning of two phosphatebinding regions, viz. 6-17 and 55-70, with respect to one another, would be identical
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to the relationship of the corresponding regions ofEF-Tu (residues 14-25 and 78-93, respectively) and ADK (residues 11-22 and 92-107, respectively). We now find in our constructed p21 model that both the relative positions and conformations of the segments are quite similar to one another as we reported for our previously constructed model (see ref 6 for a comparison of the structures). The region of p21 from residues 61-70 has the highest temperature factor in the protein (14-17), and the region from residues 59-69 in the x-ray crystallographic structure (16) was not well-defined due to its high mobility. In the deposited coordinates for p21 in the PDB, no a-carbon coordinates for residues 61-65 were determined due to the low resolution of the residues in this region although these residues are included in the published a-carbon trace (16). In previous calculations of the lowest energy structures for the p21 segment of residues 55-67 (28), we found that residues 62-67 formed an a-helix and that residues 55-58 were extended. A bend was found to occur at residues 59-60 and G1n 61 adopted the C7 equatorial conformation (28). This conformation was found to be identical to the corresponding regions ofEF-Tu and ADK (6). In the reconstructed structure of the p21 protein, the sequence from 55-58 is extended while the segment from 62-67 is helical, in agreement with the previous prediction (6).
Orientation ofthe Side Chain of Gin 61 In computing the structure of the Gin 61 region, use was made of the homology between residues 52-63 of p21 and 75-86 of EF-Tu. In computing the low-energy positions for the side chains of Gin 61, for an initial structure, it was assumed that the side chain atoms ofGln would be superimposable on the corresponding atoms ofHis 84. Energy minimization of the position of this side chain resulted in a stable conformation in which this side chain points away from the ~-phosphate ofGDP. In thex-raystructureofp21 complexed with GTP,itwas reported thatthis side chain actually extends into the phosphate-binding pocket (14,15). Dynamic simulations carried out on the computed lowest-energy conformation of the Gln 61 side chain resulted in several low-energy conformations in which the side chain points into the phosphate-binding pocket. It has been surmised that the -NH2 group of this residue may be important in the catalytic mechanism possibly by interacting with a nucleophilic water molecule in the active site (14,15). This mechanism cannot be a general one, however, because Pro and Glu can both substitute for Gin 61 without resulting in an oncogenic protein (43). Neither of these latter two residues contains a side chainNH 2 group. It is therefore unclear as to the exact role of this residue and to the exact positioning of its side chain. These considerations indicate that there are likely many low energy conformations for the side chains of amino acids in proteins - especially for polar residues on the surface. The above results also suggest that modeling of protein structure on the basis of homology can give different side chain positions from those seen in the xray crystal structure, and it is likely that both the computed and x-ray conformations are available to the side chain of Gin 61.
ras-gene Encoded p21 Protein
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Effects of Substitutions at Positions 59 It was shown in a preceding publication (27), that substitution ofThr for Ala 59 in the predicted structure would place the y-OH group of the Thr residue so that it would point into the phosphate binding pocket and would be aligned with the yphosphate ofGTP bound to the protein. Viral forms of the p21 protein contain this substitution and are known to undergo autophosphorylation by GTP (1). This alignment of the Thr residue is preserved in the current model, explaining how this residue can undergo phosphorylation by GTP in the autophosphorylation process.
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Conformation of the Effector Loop Region Residues 32-40 ofp21 have been implicated in interacting with GAP when the protein binds GTP (7). As the result of previous calculations on the structure of activated p21 proteins (27), the region from residues 35-47 was found to move significantly. The p21 peptide from 35-47 was synthesized recently and found to block oncogenic Val 12-p21 protein-induced maturation of oocytes (30). Control peptides of similar chain length were found not to inhibit the ras-effect, however. The structure of the peptide from 35-47 is shown in Figure 6. It may be noted that the conformation is an extended one. There appears to be a segregation of charges into two negative charges (Glu 37 and Asp 38; shown in red) and two positive charges (Arg 41 and Lys 42; shown in blue). The charged region is followed by a discrete hydrophobic region consisting of Val 44, Val 45, and Ile 46 (shown in green). The segregated charges and hydrophobic regions in the extended conformation may be important in the binding of p21 to intracellular effector proteins.
Limitations of the Method While the methods used in construction of the whole protein from the a-carbon trace result in the construction of a reasonable structure, there are limitations to this methodology. First, the effects of solvation have been omitted from these calculations. Solvation has been shown to be critical in the folding of the p21 protein and even for isolated segments of this protein (28). However, the modeling process was subjected to two general sets of requirements that allow exclusion of the effects of solvation: the a-carbon coordinates were required to remain close to those of the x-ray structure (except for the isolated short segment, residues 60-65 for which no a-carbon coordinates were available), and residues homologous to those in EF-Tu, a protein of very similar structure to p21 (20), were required to adopt similar conformations. Previous calculations based purely on sequence homology without even the first set of requirements resulted in successful prediction of the structure of a protein from its homologue of known structure (23) without consideration of the effects of hydration. Second, construction of the structure of p21 was based largely on its homology with EF-Tu. While there is overall forty percent sequence homology and while there is considerable structural homology between the two proteins, there are significant differences between the two proteins. For example, in structurally conserved regions,
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there are significant differences in sequence. In the phosphate-binding loop consensus sequence, the corresponding sequences for both proteins are: Gly-Ala-Gly-Gly-Val-Gly-Lys (10-16, p21)
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Gly-His-Val-Asp-His-Gly-Lys (18-24, EF-Tu) Moreover, as noted in the previous section, modelling of the side chain ofGln 61 on the basis of the conformation of its structural homologue, His 84, in EF-Tu, resulted in its pointing away from the phosphate binding site. Modeling of the side chains of p21 from the corresponding side chains ofEF-Tu in these regions is less valid than in regions where the sequence homology is greater. In these regions, therefore, the restraints holding the positions of the side chains to those of the corresponding side chains ofEF-Tu were reduced in the molecular dynamics and energy minimization procedures. In addition, the constraints involving the a-carbon coordinates tend to limit the number of possible side chain conformations for given backbone conformations. Also for the side chain of Gin 61, it is probable that this side chain can adopt a number oflow energy conformations, and it is apparently not necessary for its side chain to point into the active site as discussed in the preceding section. Third, the use of two sets of potentials, from DISCOVER and ECEPP, may conceivably lead to incompatibilities in results. In a recent comparison of potential functions used to compute peptide and protein structure (40), it was shown that three different sets of potential functions could result in major differences in the results of computing peptide structure. ECEPP was found to reproduce the single residue minima and also to predict the root-mean square end-to-end distance for poly-Ala most accurately (40). While DISCOVER was not included in this test of potentials, this program allows for bond angle bending and bond stretching which was shown to distort the (, 'I') map using the other potential functions (40). Because of this distortion, the C-7 axial conformation was found to be a low energy one with the potential functions tested (other than ECEPP) despite the fact that it is only rarely observed in protein structures (40) and is predicted to be energetically forbidden by ECEPP which does not allow distortion of bond angles and bond lengths. However, in our calculations, individual residues were required to adopt low energy conformations as defined by ECEPP. This process resulted in placement of residues like Arg 123, formerly in a high energy region, into one of the low energy single residue minima. The main use of DISCOVER was to generate representative samplings oflow energy minima for peptide segments (or side chains) that were already subjected to the constraints described above and in the Methods section. Fourth, there may be inaccuracies in the coordinates from the x-ray structure especially in the 60-65 region. The entire region from residue 59-69 is not welldefined due to high thermal fluctuations (14-17). Thus the given a-carbon coordinates in the entire region may not be as accurate as for other regions of the protein. However, the corresponding region ofEF-Tu is better defined crystallographically (25) so that homology of the residues ofp21 in this region to those ofEF-Tu allows for construction of more accurate coordinates for these residues. Nonetheless,
ras·gene Encoded p21 Protein
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further refinement of this region is being undertaken and will include the effect of solvation to determine a more accurate structure. Our coordinates for all of the atoms of residues 1-171 ofp21 complexed with GOP have been submitted to the Brookhaven National Laboratory Protein Data Base.
Acknowledgement This work was supported in part by an American Heart Association Fellowship to DCD, and NIH Grant CA42500 to MRP.
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References and Footnotes
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27. Chen, J.M., Lee, G., Murphy, R.B., Carty, R.P., Brandt-Rauf, P.W., Friedman, E., and Pincus, M.R.,J Biomol. Struct. Dynamics 6, 859-875 (1988). 28. Pincus, M.R., Brandt-Rauf, P.W., Gibson, K, Carty, R.P., Lubowsky, J., Avitable, M. and Scheraga, H.A, Proc. Nat/. Acad. Sci. USA 89, 8375-8379 (1987). 29. Vogel, V.S., Dixon, R.AF., Schaber, M.D., Diehl, R.E., Marshall, M.S., Scolnick, E.M., Sigal, I.S., and Gibbs, J.B., Nature (London) 335, 90-93 (1988). 30. Chung, D.L., Brandt-Rauf, P.W., Murphy, R.B., Nishimura, S., Yamaizumi, Z., Weinstein, I.B., and Pincus, M.R.,Anticancer Res. 11, 1373-1378 (1991). 31. Clanton, D.J., Lu, Y., Blair, D.G., and Shih, T.Y., Mol. Cell. Bioi. 7, 3092-3097 (1987). 32. Neal, S.E., Eccleston, J.F., and Webb, M.R., Proc. Nat/. Acad. Sci. USA 87,3562-3565 (1990). 33. Nemethy, G., Pottle, M.S. and Scheraga, H.A,.J. Phys. Chern. 87, 1883-1887 (1983). 34. Zimmerman, S.S., Pottle, M.S., Nemethy, G., and Scheraga, H.A,Macromolecules 10, 1-9 (1977). 35. Vasquez, M., Nemethy, G., and Scheraga, H.A, Macromolecules 16, 1043-1049 (1983). 36. Halliday, KR., J. Cyclic Nucleotide and Protein Phosphorylation Research 9, 435-448 (1983). 37. Valencia, A, Kjelgaard, M., Pai, E.F., and Sander, C., Proc. Natl. Acad. Sci. USA 88, 5443-5447 (1991). 38. Glasser, L. and Scheraga, H.A, J. Mol. Bioi. 199, 513-524 (1988). 39. Brunger,AT.,Clore,G.M.,Gronenborn,AM., and Karplus,M.,Proc.Natl.Acad. Sci. USA 83,38013805 (1986). 40. Roterman, I.K., Lambert, M. H., Gibson, K.D., and Scheraga, H.A,J. Biomol. Struct. Dynamics 7, 421-453 (1989). 41. Willumsen, B.M., Vass, W.C., Velu, T.J., Papageorge, AG., Schiller, J.T., and Lowy, D.R.,Moi Cell Bioi. 11, 6026-6033 (1991 ). 42. Jurnak, F., Science 230, 32-36 (1985). 43. Der, C.J., Finkel, T., and Cooper, G.M., Ce1144, 167-176 (1986). 44. Chen, J.M., Lee, G., Brandt-Rauf, P.W., Murphy, R.B., Rackovsky, S. and Pincus, M.R.,J. Protein Chern. 9, 543-547 (1990).
Date Received: January 20, 1992
Communicated by the Editor R.H. Sarma
