JOHNSON, CHRISTIAN WILLIAM. Structural Analysis of Arginine 97 Mutants in the Allosteric Switch of Ras. (Under the direction of Dr. Carla Mattos.)
Ras is a well-‐studied oncogenic protein involved in the Ras/Raf/Mek/Erk signaling cascade. Recent investigations using X-‐ray crystallography and in vivo
experimentation suggest that Ras-‐Raf interactions are regulated by an allosteric switch mechanism that controls the timing of their association. The allosteric switch is
by
Christian William Johnson
A thesis submitted to the Graduate Faculty of North Carolina State University
in partial fulfillment of the requirements for the degree of
Master of Science
Biochemistry
Raleigh, North Carolina 2012
APPROVED BY:
_______________________________ ______________________________
Dr. Carla Mattos Dr. Stuart Maxwell Committee Chair
DEDICATION
BIOGRAPHY How all one common weft contrives,
Each in the other works and thrives! How heavenly forces rising and descending Pass golden ewers in exchange unending, On wings with blessing fragrant
From Heaven the earth pervading,
Fill all the world with harmonies vagrant! (447-‐453; Goethe 14)
I was born April 27th 1986 at Frankford Torresdale in Philadelphia to Dr. William
Johnson and Amy Johnson. Until my second year of high school, I lived in New Hope, Pennsylvania, attending school in the New Hope Solebury school system with my two siblings: Corinne and Ryan. I trace my interest in Molecular Biology to a biology course taught by Professor Terapchik during my freshman year of high school. Our
conversations triggered my first independent thoughts of cells, chemistry, and
evolution. From this point on, the thought of working in the sciences would always stay in the back of my mind.
the University of North Carolina at Asheville (UNCA), alongside courses in chemistry and biology. However, one event would change my steering and plunge me into biology.
One night after a musical performance, I met a young woman named Christine Haynes. Her presence gave me focus, and her aspirations challenged me to consider my own passions. At the end of my sophomore year I ended my musical pursuits, realizing a real desire for scientific inquiry, and applied myself more fully to biology and
chemistry. This decision to embrace the sciences led me to my first mentor: Dr. Jennifer Ward.
I consider my time with Dr. Ward at UNCA formative to my understanding of science and the nature of life. My coursework at UNCA focused on cellular and
molecular biology, but Dr. Ward’s work involved the effects phenotypic plasticity. This divergence in study gave me a unique vantage point to view nature, from its controlled biochemistry to its greater form and variation. I conducted independent research on Piriqueta cistoides caroliniana under her tutelage for two and a half years. I performed field studies along the highway roadsides of inland Florida, cataloguing the remarkable variation in leaf morphology of P. caroliniana; I performed cloning experiments in the green house of UNCA to measure the adaptive effects of leaf variation; and I designed microarray studies to assess transcriptional variation. This last inquiry piqued my interest in the structure and function of RNA, and I applied to graduate school with the intention of becoming an RNA biologist.
and laboratory pursuits turned my interests toward protein structure and x-‐ray crystallography. I joined the lab of Dr. Carla Mattos to study the Ras protein. I became quickly enthused by the bridging of Ras structure to its role in cancer. Thus when asked, I joined Dr. Mattos and the rest of her lab in their move from NCSU to Northeastern University (NEU) in Boston Massachusetts, deciding to obtain a Masters degree from NCSU, and continuing my Doctoral studies at NEU. Christine and I married on
November 19th 2011, and moved along with our two cats, Athena and Fiona, to Boston
ACKNOWLEDGMENTS
I would like to thank all the past and present Mattos Lab members, as well as the whole Molecular and Structural Biochemistry Department faculty and staff at NCSU, for their support and encouragement. I would like to express a special thanks to Dr. Greg Buhrman and Dr. Paul Swartz for their excellent guidance while I learned the
techniques and methods required of a protein crystallographer. I would also like to thank Sue Fetics for her patience of my random laboratory questions during those early hours and weekend workdays; as well as Bradley Kearney and Mychal Smith whose advice during the solving of my crystal structures was indispensible, and especially Brad for his help in the statistics of table 3.2.1. I would also like to thank the
Brown/Hernandez lab for allowing me unrestricted use of their thermal cyclers, and the Rose lab giving me access to their purified water. Finally, I would like to especially thank Dr. Carla Mattos for her support during our move to Boston, her impetus and financial support for this Masters degree, and the time she took to edit and improve this manuscript.
I need to also thank Kathleen Davis for making the R97A mutants and solving the R97A-‐ON structure. Crystal data was collected at Southeast Regional Collaborative Access Team (SER-‐CAT) 22-‐ID (or 22-‐BM) beamline at the Advanced Photon Source, Argonne National Laboratory. Supporting institutions may be found at www.ser-‐ cat.org/members.html. Use of the Advanced Photon Source was supported by the U. S.
TABLE OF CONTENTS
LIST OF TABLES... x
LIST OF FIGURES...xi
LIST OF ABBREVIATIONS...xii
CHAPTER 1: INTRODUCTION...1
1.1Ras Biology...1
1.2Ras isoforms and argument for structural redundancy...3
1.3Mechanism of Ras activation and regulation ...4
1.4Ras structure and catalysis ...7
1.5Allosteric modulation of Ras ... 11
1.6Experimental design and hypothesis ... 15
CHAPTER 2: EXPERIMENTAL METHODS... 17
2.1 Site-‐directed mutagenesis... 17
2.2 DH5α transformation, culture and amplification ... 18
2.3 Transformation of BL21 cells and glycerol storage... 19
2.4 Expression of mutant H-‐Ras... 20
2.5 Purification of mutant H-‐Ras ... 21
2.6 SDS-‐PAGE ... 23
2.7 Ligand exchange... 23
2.8 Crystallization of R97F ... 24
2.9 Data collection, processing, and structure refinement... 25
3.1 R97F mutagenesis, expression and purification ... 28
3.2 X-‐ray data collection and results of refinement ... 28
3.3 Structure analysis of the allosteric site and network I... 28
3.4 Analysis of network II and switch II ... 36
3.5 The active site... 40
CHAPTER 4: DISCUSSION... 43
4.1 The role of arginine 97 in the allosteric site... 43
4.2 The role of calcium acetate in establishing the allosteric ON conformation ... 44
4.3 Conclusions and future directions... 49
REFERENCES... 51
APPENDIX... 55
LIST OF TABLES
Table 3.2.1: Data collection, refinement statistics, and state of allosteric
LIST OF FIGURES
Figure 1.1.1: Ras as a centerpiece in oncogenic signaling...2
Figure 1.1.2: The Ras/Raf/Mek/Erk signaling pathway...5
Figure 1.3.1: The functional GTPase cycle of Ras...8
Figure 1.4.1: Intrinsic hydrolysis of Ras... 12
Figure 1.5.1: The allosteric switch... 13
Figure 2.1.1: Site-‐directed mutagenesis of wild-‐type H-‐Ras ... 17
Figure 3.2.1: R97F crystals... 29
Figure 3.3.1: Overlay of R97 mutants with wild-‐type Ras... 34
Figure 3.3.2: Comparison of alanine 97 to arginine 97 ... 35
Figure 3.3.3: Allosteric site of R97A-‐ON and 3K8Y ... 35
Figure 3.3.4: Water interactions in allosteric region of R97A-‐ON and 3K8Y... 37
Figure 3.3.5: Hydrophobic core of R97F ... 38
Figure 3.3.6: Effect of Y137-‐water interaction in helix 3 and 4 of Ras ... 39
Figure 3.3.7: Water-‐network in R97F ... 40
LIST OF ABBREVIATIONS BLAST: Basic local alignment sequence tool
DMSO: Dimethyl Sulfoxide DNA: Deoxyribonucleic acid dNTP: Deoxyribonucleotide DTE: Dithioerythritol DTT: Dithiothrietol
EGF: Epidermal growth factor
EGFR: Epidermal growth factor receptor ExPASy: Expert protein analysis system ERK: Extracellular-‐signal-‐regulated kinase FPLC: Fast Protein Liquid Chromatography GAP: GTPase activating protein
GDI: Guanine nucleotide dissociation inhibitor GDP: Guanosine-‐5’-‐diphosphate
GEF: Guanine nucleotide exchange protein GppNHp: 5’-‐Guanylyl imidodiphosphate Grb2: Growth factor receptor bound protein-‐2 GTP: Guanosine-‐5’-‐triphosphate
HVR: Hyper variable region
IPTG: Isopropyl-‐β-‐D-‐thiogalactoside
LB: Luria-‐Bertani MW: Molecular Weight
MWCO: Molecular weight cut-‐off
MSCS: Multiple solvent crystal structures NaCl: Sodium chloride
NCBI: National Center for Biotechnology Information NCSU: North Carolina State University in Raleigh NEU: Northeastern University
OD600: Optical density at 600 nm PEG: Polyethylene glycol
Rpm: Revolutions per minute RTK: Receptor tyrosine kinase
SDS-PAGE: Sodium dodecyl sulfate polyacrylimide electrophoresis SIB: Swiss institute of bioinformatics
CHAPTER 1: INTRODUCTION 1.1 Ras biology
Research over the past few decades has revealed Ras to be an integral protein in our understanding of both cancer and the biology of intracellular signaling (Weinberg and Karnoub 2008; Pylayeva-‐Gupta, Grabocka, and Bar-‐Sagi 2011). A recent review by Pylayeva-‐Gupta, Grabocka, and Bar-‐Sagi in 2011 places Ras as a central component in the transformation of cells to a physiologically dysregulated state, encompassing cell proliferative capacity, apoptotic suppression, metabolism, microenvironment
remodeling and angiogenesis, evasion of the immune system, and metastasis. An example of the centrality of Ras in the aforementioned pathways can be seen in figure 1.1.1. Many of the roles played by Ras in oncogenesis, and likewise in normal signaling biology, have only recently been discovered. The ability of Ras to influence the
Figure 1.1.1: Ras as a centerpiece in oncogenic signaling. Note the branching and integrating role of Ras, highlighted in the diagram by a red box. Taken from Hanahan and Weinberg 2011.
phosphorylated intracellular domains of EGFR are next recognized by various adaptor proteins, such as growth factor receptor bound protein-‐2 (Grb2) and son of sevenless (SOS), that form a bridge between the activated receptor and Ras. This complex
kinase cascade that involves phosphorylation and subsequent activation of mitogen activated protein kinase kinase, called MEK. MEK acts to phosphorylate extracellular-‐ signal-‐regulated kinase (ERK), triggering ERK activation. At this point, the signaling branches off, by ERK acting to phosphorylate various transcription factors directly, or other kinases whose end target are various transcription factors. The end result of this kinase cascade is a global change in the expression patterns of the cell, tailoring and adapting the cell’s protein cohort to survive in its current environment.
1.2 Ras isoforms and argument for structural redundancy
Ras interactions with upstream and downstream effector molecules can be further complicated by the presence of Ras isoforms that are actively expressed in most cells. Ras exhibits three canonical isoforms in the adult human cell (Karnoub and
Weinberg 2008; Pylayeva-‐Gupta, Grabocka, and Bar-‐Sagi 2011): H-‐Ras, N-‐Ras, and K-‐ Ras4B. Each Ras isoform shows its own distinct pattern of oncogenic tendencies when mutated, and these largely result from differences in intracellular localization
(extensively reviewed by Castellano and Santos 2011). These differences in localization are the result of an interesting pattern of residue conservation between the three isoforms. Collectively, these isoforms retain an ~80% identity in sequence between each other, but much of the variation that does occur between them is the result of the C-‐terminal hyper variable region (HVR).
The HVR is the major site of post-‐translational processing, resulting in the
(Choy et al. 1999; Rocks et al. 2005). These modifications result in different interactions between intra-‐membrane organelles and regions of the plasma membrane; altering and limiting the interactions possible between Ras isoforms and effectors (Karnoub and Weinberg 2008).
However, the current study is focused on the G-‐protein core of Ras (residues ~1-‐ 166), which in fact exhibits a high degree of similarity (90-‐100%) between the Ras isoforms (Buhrman et al. 2011). This sequence conservation provides a rationale that analysis in the G-‐protein core of one isoform should be applicable to the others. This is supported by similar global dynamics in H-‐Ras and K-‐Ras observed by NMR (O’Connor et al, 2008; Buhrman et al 2011) as well as by identical binding site hot spot locations in the two isoforms (Buhrman et al 2011). Thus, the H-‐Ras construct used for structure analysis by me in the current study, and observations derived from it, are considered relevant to all Ras isoforms. Therefore unless appropriate, H-‐Ras will be referred to as Ras.
1.3 Mechanism of Ras activation and regulation
The function of Ras centers on its ability to cycle between an active signal transducing state, and an inactive, signaling incompetent state. This cycle is produced by the protein molecule’s nucleotide bound state (Sprang 1997; Weinberg and Karnoub 2008; Wittinghofer and Vetter 2011), where active Ras is bound to guanosine-‐5’-‐
nucleotide exchange factor (GEF). GEF’s, exampled by Sos in figure 1.1.2, represent a family of proteins that induce the release of GDP from the nucleotide binding pocket on Ras (Weinberg and Karnoub 2008). The binding of the GTP, abundant in the cytoplasm, produces a conformational change in the N-‐terminal lobe of Ras, a region that mediates effector protein interactions, including GEF interactions and GTPase activating protein (GAP) interactions.
It is in this activated, GTP-‐bound, state that Ras takes on its signaling capacity, interacting with effectors such as PI3K (Pacold et al. 2000) and Raf (Thapar, Williams, and Campbell 2004) for continued signal propagation. However, it is the temporal regulation of these Ras-‐effector interactions that can differentiate between a healthy, or pre-‐cancerous, cell. Thus suppression of Ras signaling, and subsequent inactivation of Ras, is accomplished in two ways: either by intrinsic hydrolysis (discussed in section 1.4) or through interactions with members of the GAP family of proteins. GAP’s act to increase, by at least 1000 fold (Bernards 2003), the rate at which GTP-‐bound Ras is hydrolyzed to GDP. A return to its GDP-‐bound form returns Ras to a signaling incompetent state, unable to interact with its effector molecules.
1.4 Ras structure and catalysis
The above description presents Ras as a binary switch acting at the cell membrane, with a simple ‘on’ and ‘off’ state dependent on the binding of a guanine nucleotide. But this simple model hardly describes the complex conformational changes involved at the GDP/GTP binding site of Ras, nor the mechanism of hydrolysis.
figure 1.3.1, or intrinsic hydrolysis. Early studies produced conflicting models of
hydrolysis. Work done by Scheffzek et al. in 1997 argued for an associative mechanism based on the transition state mimic Ras-‐GDP-‐AlF3/RasGAP complex in which arginine
789 of GAP, the so-‐called ‘arginine finger’, is found at the active site of Ras near the γ-‐
phosphate of GTP. This would be consistent with stabilization of negative charges accumulating at the γ-‐phosphate oxygen atoms during an associative transition state. In
contrast, evidence from studies involving enzyme-‐free hydrolysis of GTP supported a dissociative-‐like reaction, involving the accumulation of negative charge at the bridging oxygen atom between the β-‐ and γ-‐phosphates of GTP (Maegley, Admiraal, and
both GAP-‐catalyzed and intrinsic hydrolysis occurs through a loose transition state, characteristic of a dissociative mechanism (Du et al. 2004; Du and Sprang 2009).
GAP plays a second important role in increasing the rate of GTP hydrolysis. GAP binding occurs at the ‘effector’ lobe, the region of Ras that includes switch I (residues 30-‐40), switch II (60-‐76), and the P-‐loop (residues 10-‐17) surrounding the nucleotide-‐ binding site. In the unbound state, the switch I and II regions of Ras-‐GppNHp (a GTP analogue) are in a disordered state (Geyer et al. 1996). Binding of GAP produces a viable catalytic site by ordering switch I and II near the nucleotide, a process that is necessary for catalysis in that it brings important residues into interaction with the β-‐
and γ-‐phosphates. For switch I and II, these interactions include the backbone carbonyl
group of threonine 35 and the δ-‐oxygen atom and backbone amide of glutamine 61. In
the P-‐loop, the amide of glycine 12 interacts with the γ-‐phosphate of GTP.
The structure of Ras-‐GppNHp solved from a crystal with symmetry of space group P3221 suggested a two-‐water mechanism of hydrolysis involving substrate-‐
assisted catalysis (Scheidig, Burmester, and Goody 1999; Buhrman et al. 2010). This model involves γ-‐phosphate abstracting a proton from a nearby water molecule that
subsequently activates a catalytic water molecule for nucleophilic attack on the γ-‐
31P NMR studies (Geyer et al. 1996) suggested that intrinsic hydrolysis occurs with
tyrosine 32 closed over the nucleotide, a conclusion that could not have been inferred from the originally solved structure with P3221 symmetry.
The NMR experiments by Geyer et al. in 1996 showed that switch I, in solution, samples two stable conformers, state 1 and state 2. Further, these experiments show that each conformer of switch I is exclusively stabilized when either bound to GAP (state 1) or Raf (state 2), an especially intriguing result considering that Raf only binds at switch I, unlike GAP which binds both regions (Scheffzek et al. 1997; Buhrman, Wink, and mattos 2007). In 1996, Nassar et al. solved the structure of Raps, a homolog of Ras mutated to have the Ras-‐switch I sequence, bound to a GTP analogue GppNHp, in complex with the RBD (Ras-‐binding domain) of Raf. Their structure of the Raps/Raf-‐ RBD complex revealed the structure of switch I in state 2. It shows tyrosine 32, a
conserved residue in the Ras GTPase family, to be in a conformation very different from that seen in the Ras/RasGAP complex. In the complex with GAP, tyrosine 32 is flipped away from the GTP analogue, while in the Raps/Raf-‐RBD structure, tyrosine is seen lying over the β-‐ and γ-‐phosphates, interacting with a water molecule that bridges its
hydroxyl group to the γ-‐phosphate of GppNHp, (termed the bridging water molecule).
These differences in conformation of tyrosine 32, combined with the NMR studies above, suggested that intrinsic hydrolysis was occurring by a different mechanism than the two water substrate-‐assisted models presented above.
2007; Buhrman et al 2010). The P212121 space group of the Raps/Raf-‐RBD complex has
switch II in crystal contacts, presenting a problem for verifying the correct switch II conformation of Ras required for intrinsic hydrolysis. The R32 space group in contrast leaves switch II untouched by crystal contacts, while its switch I region is in crystal contacts that stabilize its conformation to that observed in the Raf-‐Raps interaction. Using the R32 space group and wild-‐type Ras-‐GppNHp, Buhrman, Wink and Mattos in 2007 presented a new model for intrinsic hydrolysis. This mechanism of intrinsic hydrolysis (figure 1.4.1) relies on a proton being transferred from the catalytic water molecule, through the γ-‐phosphate, to tyrosine 32, glutamine 61, and a bridging water
molecule. This proton serves to stabilize the build up of negative charge on the bridging oxygen between β-‐ and γ-‐phosphate in the transition state, where it is then delivered to
the GDP leaving group (Buhrman, Wink, and Mattos 2007; Buhrman et al. 2010). 1.5 Allosteric modulation of Ras
By crystallizing Ras-‐GppNHp in the presence of calcium acetate, and by soaking in calcium acetate, Buhrman et al. were able to show that a calcium ion and an acetate molecule induce a change in the conformation of Ras that results in the ordering of switch II (figure 1.5.1). These changes occur in the global scale of the molecule, reaching from the remote allosteric binding site all the way to the active site of Ras. These
Binding of calcium acetate at the allosteric site is mediated by key amino acid residues in helix 3, loop 7 and helix 4 (Buhrman et al 2010). Collectively, residues within this site participate in a network of hydrogen bonding interactions that result in helix 3 shifting toward helix 4. This shift of helix 3 provides the necessary space for the
C-‐terminal end of switch II to take on a α-‐helix conformation, leading to a conformation
at the N-‐terminal end of switch II that is highly ordered around the active site. Two hydrogen-‐bonding networks relay this allosteric change in conformation. Network I is centered at the allosteric site as already mentioned, and involves the bound calcium acetate. Tyrosine 137 (helix 4) makes a hydrogen bond to histidine 94 (helix 3), forming the base of the site. Lysine 101, glutamate 98, and arginine 97 (helix 3) form a series of interactions that lead to a salt bridge interaction between arginine 97 and the acetate molecule. Finally, the backbone carbonyl group of aspartate 107 (loop 7), the carbonyl group of tyrosine 137, one of the acetate oxygen atoms and three water molecules coordinate the calcium ion forming a typical hexa-‐coordination sphere. On the opposite side of helix 3, glutamine 99 is the start of network II, a dense hydrogen-‐bonding
network that includes several water molecules. Arginine 68 (switch II) is at the center of this network, its side chain interacting with four water molecules, which in turn hydrogen bond and coordinate tyrosine 96, glutamine 99, serine 65, and glutamate 62. Ordering of switch II then allows glutamine 61 to take up its proper position for
catalysis (figure 1.4.1).
Analysis of the allosteric ON and OFF wild-‐type Ras-‐GppNHp structures, in light of the R32 space group, suggested a model for the regulation of Ras in the
timing of Ras-‐Raf interaction, and thus regulation of the Ras/Raf/Mek/Erk pathway, is controlled by the allosteric switch, where first Raf binding, and then a second binding event in the allosteric site results in ordering of the catalytic site, increasing the rate of GTP hydrolysis. This model explains a paradox of intrinsic hydrolysis, where in vivo rates of pathway activation (Buhrman et al. 2011) and in vitro rates of intrinsic hydrolysis are not comparable (GTP t1/2=~55 minutes, unpublished data). Consistent
with this model is the binding affinities of Raf compared to other Ras effectors. Raf shows a 103 fold higher affinity for Ras (3.5 nM; Buhrman et al. 2010; Minato et al.
1994) compared to GAP, PI3K, and RalGDS (Vogel et al. 1988; Pacold et al. 2000; Herrmann et al 1996; Buhrman et al. 2010). This raises the question of whether GAP is able to displace Ras-‐bound Raf in order to activate hydrolysis leading to suppression of signaling down the Raf/Mek/Erk pathway.
1.6 Experimental design and hypothesis
phenylalanine 97 to create stacking interactions with the ring of tyrosine 137, an interaction that occurs with arginine 97 when Ras is in the allosteric ON conformation, pulling helix 3 toward helix 4. An arginine 97 to alanine mutation using site-‐directed mutagenesis, crystallized under calcium acetate enriched conditions and different temperatures, was performed by Kathleen Davis and used for comparative analysis. This alanine mutation mimics the loss of charge at the allosteric site and effectively removes the side chain at position 97, while still maintaining the chirality and φ/ψ
limits required for the α-‐helical conformation of helix 3. The effect of these mutations is
then determined by comparing wild-‐type H-‐Ras structures in their allosteric ON (PDB: 3K8Y) and OFF (PDB: 2RGE) conformations.
CHAPTER 2: EXPERIMNETAL METHODS 2.1 Site-Directed Mutagenesis
Site-‐directed mutagenesis experiments were performed using an ampicillin selective pET DNA plasmid vector containing codons 1-‐166 of wild-‐type H-‐ras, with
two minutes at 58.3˚C, and primer extension for five minutes at 68˚C. Mutagenesis of arginine 97 to phenylalanine was performed in a 51 µL solution containing 5 µL of 10X
cloned Pfu reaction buffer (Agilent), 1.25 µL (1 ng) each of forward and reverse
primers, 1 µL of dNTPs (New England BioLabs), 1 µL of DMSO, 2 µL of pET vector of H-‐
Ras (10 ng/µL) and 38.5 µL of sterilized and purified water. Just prior to thermal
cycling, 1 µL of PfuTurbo® Hotstart DNA polymerase was added to the reaction solution.
Post-‐thermal cycling, the reaction was thoroughly mixed with 1 µL of DpnI restriction
enzyme (New England BioLabs) then briefly centrifuged, and incubated at 37˚C for 60 minutes.
2.2 DH5α transformation, culture and amplification
Amplification of mutagenized plasmids was performed by the transformation of DH5α™ competent Escherichia coli cells by the arginine 97 to phenylalanine mutant
plasmid (hereafter called the R97F for short). DH5α™ transformation was accomplished
by addition of 5 µL of mutagenesis product to 100 µL of thawed DH5α™ competent
cells, and incubated for 30 minutes on ice. Immediately following incubation, DH5α™
cells were heat shocked for 30 seconds at 42˚C to induce uptake of mutagenized plasmid. DH5α™ cells were then placed back on ice for up to two minutes, and then
mixed with 250 µL of pre-‐warmed SOC media. Incubation of DH5α™ was accomplished
at 37˚C at 225 rpm for 3 hours.
Post-‐incubation, 100 µL and 200 µL from each transformation was plated using
at 37˚C. Colonies from overnight culture were picked using a pipette tip, and used to inoculate 10 mL of LB broth and ampicillin (1mg/20 ml). Inoculated broth was then incubated overnight at 37˚C and 225 rpm.
Amplified DNA was extracted and purified from DH5α™ cells using the QIAprep®
Miniprep protocol (Qiagen) with limited modification. Approximately 3 mL from the overnight LB broth and ampicillin culture was used to form a cell pellet by
centrifugation at 10,000 rpm. DH5α™ cells were re-‐suspended and lysed using QIAprep®
Miniprep supplied buffers. QIAprep spin columns provided in the kit were used to purify and suspend plasmid DNA in 10 mM Tris pH 8.5 to a final volume of 50 µL.
Mutagenesis of arginine 97 to phenylalanine in the H-‐Ras sequence was
validated by third-‐party DNA sequencing. 10 µL of purified DNA was placed in a 1.5 mL
eppindorf tube and sent via overnight mail to Eurofins MWG-‐Operon. Sequence data were then returned digitally through MWG-‐Operon’s server and translated to its amino acid sequence using ExPASy Translate tool (Gastieger et al. 2003). Success of
mutagenesis was determined by using protein BLAST (Altschul et al. 1997) to compare the translated amino acid sequence to wild-‐type H-‐ras amino acid sequences in the NCBI database.
2.3 Transformation of BL21 cells and glycerol storage
described in section 2.2. Transformed BL21 cells were stored in glycerol, as described below.
Transformed BL21 cells were used to inoculate vials containing LB broth and ampicillin then incubated overnight at 37˚C and 225 rpm. After incubation, 1.5 mL of the inoculated solution was centrifuged for 25 seconds at 12,000 rpm in order to form a cell pellet. Transformed BL21 cells were subsequently re-‐suspended by gentle
vortexing in a sterile 1:1 solution of MgSO4 and 80% glycerol. BL21 glycerol stocks were
stored at -‐80˚C for later use. 2.4 Expression of mutant H-Ras
Glycerol stocks of transformed BL21 cells were used to inoculate 200 mL of sterilized LB broth and ampicillin, incubated overnight at 37˚C and 225 rpm. Post-‐ incubation, the overnight culture was divided evenly between four 1.5 L volumes of sterilized LB broth, and subsequently incubated again overnight. BL21 cell growth was determined by examining the OD600 at 60, 100, and 130 minutes. When liquid cell
cultures reached an optimal density for protein expression (0.64-‐0.87 AU at ~130 minutes), 150 mg of IPTG was added to each 1.5 L solution to induce mutant plasmid expression. Concomitant with IPTG addition, the incubation temperature was dropped to 32˚C and allowed to grow uninterrupted for five hours.
After a five-‐hour incubation, each 1.5 L was transferred centrifuged at 4˚C and 17,000 rpm using a Sorvall RC-‐5B plus (Spectrofuge corporation). Cell pellets were combined and stored in a sample cup at -‐80˚C for later use.
2.5 Purification of mutant H-Ras
The frozen cell pellet was evenly suspended in a metal sonication cup, using 100 mL of Buffer A pH 8.0 (20 mM Tris, 5 mM MgCl2, 50 mM NaCl, 5% glycerol, 1mM DTT,
20 µM GDP) containing the following peptidase inhibitors, each at 1mg/mL: leupeptin,
pepstatin, antipain, and F-‐64 (Sigma-‐Aldrich). The protein was kept on ice to hinder denaturation during all of the purification steps. Once the cell pellet was thawed and thoroughly suspended, cells were lysed by sonication. The sonication regime consisted of five cycles of 30 seconds on and 30 seconds off. Lysed cells were next divided into equal volumes and centrifuged at 4˚C and 17, 000 rpm for 20 minutes. The supernatant was decanted into a glass beaker with a magnetic stir bar and gently stirred. Nucleic acid precipitation was accomplished by slow addition of PEI to 0.02% concentration, and then allowed to stir for 20 minutes. This solution was then divided into equal volumes and centrifuged at 4˚C and 17, 000 rpm for 20 minutes. The supernatant after PEI precipitation and centrifugation was sequentially filtered using a 0.5 µm and 0.45
µm filters (Millipore) and glass fiber pre-‐filters (Millipore). All steps were performed on
ice to hinder protein denaturation.
Separation and purification of R97F from bulk protein was accomplished using the AKTA FPLC from Amersham Biosciences. Protein was first separated by anion exchange chromatography using a HiPrep™ 16/10 Q Sepharose Fast Flow column (GE Healthcare) pre-‐cleansed in Buffer B pH 8.0 (20 mM Tris, 5 mM MgCl2, 1 M NaCl, 5%
of buffer B to buffer A). Fractions from the tallest peak in the chromatogram were validated for the presence of Ras using SDS-‐PAGE (method described in section 2.6) and pooled. Pooled protein was concentrated to 5 mL using a 15 mL 30,000 MWCO Amicon®
Ultra centrifugal filter by Millipore at 14,000 rpm and 4˚C using a Sorvall Legend XTR (Thermo Scientific), and injected into a 5 mL loop for further purification.
The next stage of purification was size exclusion chromatography using a HiPrep™ 26/60 Sephacryl™ S-‐200 High Resolution column (GE Healthcare). Proteins were fractionated by their size and shape. Protein fractions were pooled based on peak location in the gel filtration chromatogram and the expected migration of Ras through the column. Fractions containing Ras protein were validated by SDS-‐PAGE.
Pooled protein was further purified using anion exchange chromatography by using a HiPrep™ 26/60 QHP column (GE Healthcare) in order to remove any residual and contaminating protein. HiPrep™ 26/60 QHP columns are a standard method to clean up contaminants in the last stages of purification. Protein fractions were pooled based on their peak location in the QHP chromatogram, and verification of Ras protein by SDS-‐PAGE. Pooled protein was immediately concentrated, as described above, to 5 mL.
A second round of size exclusion chromatography was used to further purify H-‐ Ras, as previous purifications had shown a high molecular weight contaminant. Though the current purification did not show a high MW contaminant, the protocol was
mM NaCl) was used, as it was thought that the contaminant was interacting with Ras in a binding interaction that was not removed during ion exchange chromatography. 2.6 SDS-PAGE
SDS-‐PAGE was performed for detection of R97F in fractions, as well as a qualitative assessment of fraction purity. Eight microlitre aliquots from each fraction were analyzed by mixing with 2 µL of sample buffer (50 mM Tris-‐HCL pH 6.8, 2% SDS, 10% glycerol, 1% β-‐mercaptoethanol, 12.5 mM EDTA, 0.02% bromophenol blue), and
then by heating samples for 10 minutes at 95˚C. Each round of electrophoresis included 6 µL of Precision Plus Kaleidoscope Standard (Bio-‐Rad) run on the most far left lane. Sample volumes were separated using a 15% polyacrylimide gel for 80 minutes at 100 milliamps. Visualization of protein bands was accomplished by immersion in Coomassie blue stain.
2.7 Ligand exchange
Prior to ligand exchange, purified protein was concentrated to 2 mL at a concentration of 22.3 mg/mL using the same method outlined in section 2.5. For exchange of GDP for GppNHp, 2.5 mL Illustra NAP-‐25 gravity gel filtration columns (GE Healthcare) were used to remove free GDP from concentrated protein, and later to remove excess GppNHp from exchanged samples.
Illustra NAP-‐25 columns were prepped with either reaction buffer (32 mM Tris pH 8.0, 200 mM ammonium sulfate, 10 mM DTT, 0.1% n-‐octylglucopyranoside) or stabilization buffer (20 mM Hepes pH 7.5, 20 mM MgCl2, 10 mM DTT, 50 mM NaCl).
Following equilibration, concentrated protein was first passed through the reaction buffer-‐containing column to remove free GDP. One mL volumes were taken from the elution, and presence for protein was determined using a Bradford reagent protein assay. Identified protein fractions were then pooled and allowed to incubate, in the presence of 20 µM GppNHp, with alkaline phosphatase beads under gentle rotating
conditions for 60 minutes at 37˚C. Post-‐incubation, 20 mM of MgCl2 was added to each
protein solution, and allowed to sit for 2 minutes. The solution was then centrifuged at 1000 rpm and 4˚C. The resulting supernatant was then passed through the stabilization buffer equilibrated column. Fractions were again analyzed using a Bradford protein assay. Protein was finally pooled and concentrated to 22 mg/mL, and immediately flash frozen in 50 µL aliquots of liquid nitrogen and stored at -‐80˚C for later crystallization.
2.8 Crystallization of R97F
as follows: 400 µL of PEG/Ion screen #28 from Hampton Research (0.2 M calcium
acetate hydrate, 20% w/v PEG 3,350, pH 7.5), 25 µL of stabilization buffer (formulation
in section 2.6), and 100 µL of 50% w/v PEG 3,350. Drops consisted of 2 µL of PEG/Ion
screen #28 and 2 µL of purified protein 22 mg/mL. Drops were not mixed. Crystals
grew at 18˚C for 7 days. Crystals chosen for collection were flash frozen in liquid nitrogen using a cryoprotectant consisting of 70% reservoir solution and 30% PEG 3,350 (50% w/v).
2.9 Data collection, processing, and structure refinement
X-‐ray diffraction data were collected at Argonne National Laboratories (IL) using the SER-‐CAT ID-‐22 beamline. Diffractions were obtained with an X-‐ray wavelength of 1.0 Å. Each frame was exposed for 2.5 seconds at an oscillation angle of 1˚. Data were collected on a mar345 phosphorimaging plate at a crystal to detector distance of 150 mm. Diffraction data of mutant Ras were then indexed and scaled using HKL2000 (Otwinowski and Minor, 1997).
simulated annealing within the first round of refinement started at 2500K and ended at 300K. Analysis of this initial structure (Coot, Emsley and Cowtan 2004) showed that 3K8Y was a poor starting reference structure. Ras exists in two, ON or OFF, allosteric states— with the structure of PDB code 3K8Y being an example of the ON state model. A first look at the R97F structure suggested that this structure was predominantly in the OFF state—making the structure with PDB code 2RGE, an OFF state model, a better reference structure. Refinement with simulated annealing was re-‐performed using 2RGE as a reference model, yielding a better start approximation of phases for the R97F structure. Before another round of refinement was performed, the GppNHp molecule and coordinating magnesium ions were added to the R97F structure as follows: the 2RGE structure was superimposed on the R97F structure using LSQ, a least-‐squares fitting strategy. This superimposed structure file, now containing comparable Cartesian coordinates to R97F, was edited to contain only the GppNHp molecule and its
coordinating magnesium ion; finally, the 2RGE truncated file and crude R97F structure were merged in Coot. A residue-‐by-‐residue walkthrough was then performed to better conform the R97F model structure to the calculated 2FO-‐FC and FO-‐FC maps generated
by initial refinement and simulated annealing. The 2RGE model lacks residues 61-‐68 (switch II) due to the characteristic disordered state of switch II in the allosteric OFF state, but R97F displayed a crude electron density map. Subsequent rounds of
switch II region using a similar method described above for introduction of the GppNHp and magnesium ions into the R97F structure (refinements 7-‐8). However, subsequent analysis of the switch II structure showed that addition of switch II in the R97F
structure was unnecessary, as a result of minor allosteric conformations in the R97F crystal, and was therefore removed. A final refinement of R97F was then performed.
Two initial structures of H-‐Ras containing arginine 97 to alanine mutations, hereafter called R97A, at 2.3 and 1.7 Å resolution, were supplied by Kathleen Davis (NCSU, Mattos Lab) for comparison to the R97F structure. The 2.3 Å resolution
CHAPTER 3: RESULTS 3.1 R97F mutagenesis, expression, and purification
Sequencing of R97F plasmid showed that site-‐directed mutagenesis of H-‐ras was successful, and this plasmid was used for mutant protein expression in BL21 cells. Cell pellets of R97F transformed BL21 cells were used for purification, as described in sections 2.5. Results of purification are depicted as chromatograms for anion exchange and size exclusion chromatography in figure A.1, and accompanying SDS-‐PAGE analysis of each chromatogram figure A.2. SDS-‐PAGE of the final size exclusion chromatography shows that concentrated R97F was relatively pure.
3.2 X-ray data collection and results of refinement
R97F crystals grew to 1.5-‐3 microns on their longest edge as triangular prisms (Figure 3.2.1). Crystals diffracted to 1.5 Å resolution at the SER-‐CAT ID-‐22 beamline. Data collection and refinement statistics can be found in table 3.2.1.
3.3 Structure analysis of the allosteric site and network I
an acetate molecule and a coordinated calcium ion. Replacement of arginine 97 with alanine produced little perturbation. Electron density from tyrosine 137 suggests that the removal of arginine in this hydrophobic pocket allows tyrosine 137 to shift toward alanine 97 (figure 3.3.2); a shift that is echoed in the 3K8Y model. A noticeable
difference between the allosteric sites of R97A-‐OFF and 2RGE, is that R97A-‐OFF has a significantly less hydrated allosteric site. This is not surprising given the removal of a charged side chain from the site.
sites confirms the allosteric ON conformation of R97A-‐ON. Like the R97A-‐OFF model, a shift of tyrosine 137 toward alanine 97 occurs, but unlike R97A-‐OFF, R97A-‐ON shows glutamate 98 moved into the allosteric site, as well as histidine 94 taking on two alternative conformations. One conformation of histidine 94 is at a low occupancy, as suggested by weak electron density, where it is flipped towards helix 4 and has lost its hydrogen bonding to tyrosine 137 (figure 3.3.3). The second conformation of histidine 94 is similar to that of the allosteric OFF state found in 2RGE and R97A-‐OFF. This second conformation of histidine 94 places glutamate 98 in nearly the same plane, with a hydrogen bond between ND1 of the histidine ring and an oxygen atom of the side chain of glutamate 98. This difference between R97A-‐ON and 3K8Y at histidine 94 and glutamate 98 can be attributed to changes at the hydrophobic pocket below the
and the backbone carboxyl groups of residues 137 and 138, in R97A-‐ON orient two water bridges that mimic the conformation of the allosteric site in 3K8Y.
The most apparent difference between R97A-‐ON and 3K8Y is the absence of acetate and calcium in the allosteric site (figure 3.3.4). This may at first be surprising considering the allosteric ON state of R97A-‐ON, but given that acetate interacts closely with arginine 97 in wild type it would not be expected to bind in its absence. Electron density suggests a water molecule occupies the location of calcium in R97A-‐ON, where it donates hydrogen bonds to the backbone carbonyl groups of tyrosine 137 and aspartate 107 (figure 3.3.4a). The identity of a water molecule, as compared to a
calcium ion at low occupancy, is evident for a number of reasons. First, there is no clear evidence of hexa-‐coordinating electron density, as there should be for a calcium ion. Second, the backbone carbonyl groups of aspartate 107 and tyrosine 137 form
hydrogen bonds at 2.60 Å, whereas in 3K8Y the bond lengths are 2.44 and 2.5 Å. Third, the overall conformation of the 2.3 Å R97A-‐ON structure would suggest a stronger occupancy of calcium, by displaying much stronger electron density in this region. Superposition of the R97F H-‐Ras mutant to both 3K8Y and 2RGE at the allosteric site shows that R97F is in the OFF conformation, as evident by placement of helix 3 toward switch II (figure 3.3.1c). Replacement of arginine with phenylalanine at residue 97 modifies a nearby hydrophobic pocket, producing packing interactions between tyrosine 137, isoleucine 93 and 139, and mutant residue phenylalanine 97 at the core of the protein (figure 3.3.5). These packing interactions result in a flip of side chain
137 and arginine 97 in the 2RGE model. In R97F, tyrosine 137 makes a ~90˚ flip toward the allosteric site and the phenylalanine side chain inserts itself into the hydrophobic pocket toward isoleucine 93, producing tight packing between the residues (distance measurements from phenylalanine: 3.40-‐3.50 Å to tyrosine 137; 3.48 Å to isoleucine 93; 3.70 Å to isoleucine 139; 4.11 Å to leucine 113). The shift in tyrosine 137 allows a
hydrogen bond to form between a water molecule and the side chain’s hydroxyl group (figure 3.3.6). This new interaction of tyrosine 137 causes a shift of both glutamate 98 and histidine 94 toward tyrosine 137 (compared to the 2RGE structure). Interestingly, the position of this water molecule interacting with tyrosine 137 mimics the location of an oxygen atom on the glutamate 98 side chain in 3K8Y.
stable backbone conformations of aspartate 107 and tyrosine 132 in the OFF state, argues against a calcium being placed in lieu of a water molecule. Remarkably, R97F shows conservation of a water molecule network found in 3K8Y (but not 2RGE) that hydrogen bonds to the water molecule occupying the calcium ion-‐binding site exhibited in 3K8Y (figure 3.3.7). This water-‐network connects the backbone carbonyl groups of tyrosine 137 and glycine 138 with the side chain of glutamate 162. A minor ON conformation is also seen in R97A-‐OFF, revealing an influence of arginine 97 mutants on the natural equilibrium between ON and OFF allosteric conformations.
Oddly, R97F and R97A-‐OFF show the presence of a calcium ion that forms a salt bridge in a symmetry position with the side chains of aspartate 105. The conformation of aspartate 105 in both of these structures is unique: neither 3K8Y, 2RGE, or R97A-‐ON show a similar conformation of aspartate 105, nor a calcium ion bound to its side chain. 3.4 Analysis of network II and switch II
Concomitant with the dominant allosteric OFF conformation, both R97F and R97A-‐OFF show the C-‐terminal half of switch II (residues 69-‐73) in a similar
conformation to that observed in the structure with PDB code 2RGE, with a disordered N-‐terminal half of switch II (60-‐68). However, as mentioned above both R97F and R97A-‐OFF show a minor contribution associated with the ON state of the allosteric switch, where there is a fully ordered switch II. Due to this contribution there is
electron density present for the N-‐terminal half of switch II in both structures. Since the electron density for the ON state is not sufficient to warrant the inclusion of the
out for consistency in both structures. Residues 61-‐68 were omitted from the R97A-‐OFF model and residues 62-‐68 were omitted from the R97F model. Interestingly, there is strong electron density for glutamine 61 in the R97F structure, suggesting full
occupancy for this residue, even though it is usually disordered in the OFF state of the allosteric switch.
aspartate 69 and glutamate 62. Aspartate 69 takes on a different conformation that is not seen in the R97A-‐OFF, R97F, 3K8Y, or 2RGE structure, but forms a hydrogen bond with a water molecule, which in turn forms crystal contacts with another molecule in the unit cell. The side chain of glutamate 62 is facing into the protein core, whereas in the ON conformation of 3K8Y, this residue is flipped out toward the bulk solvent. With the exception of aspartate 69, the rest of network II appears to be intact in the R97A-‐ON structure.
3.5 The active site
In keeping with the R97A-‐ON structure, the active site of this mutant
superimposes well with that of 3K8Y. Although the R97A-‐ON structure was solved at lower resolution than 3K8Y (Table 3.2.1), the location of the catalytic water molecules are supported by electron density and overlap well with their positions in the 3K8Y structure. Unlike the R97A-‐ON structure however, R97A-‐OFF and R97F mutants show a modified active site, with no bridging water molecule and a direct hydrogen bond between the hydroxyl group of Y32 and the γ-‐phosphate.
In the R97F mutant, concomitant with an absent bridging water molecule, tyrosine 32 shifts 1.49 Å toward the oxygen 1 atom of γ-‐phosphate, as well as a 0.86 Å
drop of glutamine 61, at its Cδ, toward the γ-‐phosphate of GppNHp (figure 3.5.1).
Similar interactions are observed in the R97A-‐OFF structure. The orientation of
tyrosine 32 and glutamine 61 in the R97F structure results in an active site that mimics the anti-‐catalytic conformation of the Q61L mutant (PDB code 2RGD). However,
difference electron density at tyrosine 32, more so in the R97A-‐OFF than in the R97F structure, shows an alternate conformation of this residue that places it in the position observed in the 3K8Y and 2RGE structures where the bridging water molecule is present. Interestingly, there is no evidence for a low occupancy bridging water molecule to act in conjunction with the alternate tyrosine 32 conformation.
