Lab 5: Proteins and the small molecules that love them (AKA Computer Modeling with PyMol #2)

Transcription

1 Lab 5: Proteins and the small molecules that love them (AKA Computer Modeling with PyMol #2) Goals: The objective of this lab is to provide you with an understanding of: 1. Catalysis 2. Small molecule inhibitors 3. HMG-CoA Reductase inhibitors Introduction: As you have learned in the lectures, life is dependent upon chemical reactions, such as the conversion of DNA monomers into DNA polymers; RNA monomers into mrnas, trnas and rrnas; amino acids into proteins; and the cleavage of HIV polyproteins into functional proteins. These chemical reactions are thermodynamically favorable, however if these chemical reactions were left to occur on their own, the rate would be too slow to support life. Thus the rates of the chemical reactions need to be enhanced. In the laboratory synthesis of Flexat to Diolat (Lab 5), you saw how chemical reactions can be performed outside of the cell. Reaction rates can be accelerated through the addition of heat, altering the ph of the solution, or adding catalysts a reagent(s) that speeds up a chemical reaction without being consumed. The first two options are not very readily available inside a cell. The inside of a cell cannot have it s ph altered without having deleterious consequences for many other processes and the temperature inside each cell is carefully maintained in many organisms. However catalysts are available usually in the form of enzymes. In this course, you have already been introduced to several enzymes that catalyze chemical reactions increase the rate of a chemical reaction without being consumed themselves: DNA Polymerase, RNA polymerase, and the Ribosome. Many other cellular chemical reactions are also dependent upon enzymes. The breakdown of proteins, lipids, and carbohydrates ingested as food are dependent upon enzymes. The post-translational processing of proteins often require the assistance of enzymes. The biosynthesis of small molecules is also dependent upon enzymes. These enzymes are able to catalyze chemical reactions by employing several strategies: 1. Orientation and proximity by coordinating chemical reactants in their ideal orientations and in close proximity, they can increase the rate of a chemical reaction by effectively increasing the local concentration of the substrates and placing the atoms involved in the chemical reaction in their ideal positions for the reaction; 2. Nucleophilicity and Electrophilicity through the precise positioning of amino acid sidechains, the enzyme can alter the chemical features of individual atoms in a substrate to have an enhanced affinity for positively or negatively charged reactants respectively. Enzymes have another feature that distinguish them from chemical catalysts they are extremely specific in their choice of substrates. Enzymes bind 87

2 substrates in a region of the protein referred to as the active site. The active site is where the chemical reaction will occur, but the shape and chemical features (position of hydrogen bond donors and acceptors, hydrophobic, or hydrophilic side chains) are such that only a very small subset of molecules will be able to fit. This specificity ensures that the only very specific molecules will be acted upon by any given enzyme. Because of the precise shape and features of an enzymes active site, biochemical reactions can also be inhibited by designing specific inhibitors that prevent the function of very specific enzymes. In this lab we will investigate three different enzymes and see how the specific positioning of enzyme sidechains allows for substrate specificity and increases the rate of catalysis. Moreover we will see how two enzymes, HIV Protease and HMG-CoA Reductase, can be beneficially inhibited by small molecule inihibtors. Procedures: This lab consists of six sections; each section will utilize a PDB coordinate file and an accompanying script. The questions will be due one week from the day of the lab. 88

3 Section 1: Catalysis and Trypsin Open up the folder titled 1_Catalysis, then double-click on the PyMol file labeled Trypsin.pml. This will automatically open up the PyMol program, and load up the coordinates for the DNA structure. In this section you will look at Trypsin to better understand how proteins orient specific sidechains to bind specific molecules and catalyze chemical reactions. As you learned in lecture from David, Trypsin is a key catalytic enzyme required for breaking down proteins ingested as food. If trypsin were able to digest any protein, it would be problematic as it would digest itself and once digested, unable to function. Thus trypsin must accelerate the digestion of specific proteins or peptides while sparing itself. The first image you will see is of Trypsin in stick form. Notice the overall globular nature of the protein. As a reminder, oxygen is colored red, carbon is green, and nitrogen is blue. Click off Trypsin_Sticks and click on Trypsin_Cartoon. Remember that a proteins structure often consists of multiple secondary structure elements. In this image you can clearly see α-helices and β-sheets. You will also notice many loops with less defined structures. Click on Substrate_Sticks. The substrate carbons are labeled purple to distinguish it better from Trypsin. Notice how it interacts. As you will see later, the interaction of the substrate with trypsin occurs at very specific positions, near specific residues. 1) The substrate is a folded protein, but trypsin only interacts with a portion of it on the surface. One very prominent sidechain can be seen from the substrate interacting with Trypsin. What is this amino acid? Click off Substrate_Sticks, turn on Substrate_Cartoon. Notice the region of the substrate that interacts with Trypsin. In this case it is a loop region of the substrate rather than an α-helical or β-sheet region. Turn on Cat_Triad. As you ll recall from lecture, the active site of a protein is not always formed from a continuous stretch of amino acids. The key amino acids can be quite distant in 89

4 the primary structure. In this image, three residues essential for Trypsins function are highlighted. 2) Are these three key residues positioned next to one another in the primary sequence of Trypsin? 3) Identify the three residues. The three residues that are shown are essential for hydrolysis of the peptide backbone of the substrate. Together these three amino acids are referred to as a catalytic triad. The precise positioning of these amino acids allows them to efficiently hydrolyze the peptide backbone. It turns out that the residue closest to the substrate is the one that performs the chemical reaction, however the other two are essential for stabilizing the ionic charges that form during hydrolysis. Turn on Cleavage_Site. The site of the substrate is now highlighted. The specific bond to be hydrolyzed is located between these two residues. 4) What are the two amino acids shown on the substrate? 5) Which is the more N-term residue, more C-term residue? How do you know? Turn on Asp_189. Aspartate 189 is a key residue that determines substrate specificity. As you ll recall, aspartate is an amino acid with an acidic sidechain thus at neutral ph it is often deprotonated and has a negative charge. Turn off all except Asp_189 and Cleavage_Site. 6) In this image what residues interact, and how do they interact? Turn on Binding_Pocket. Now we can see specifically how the substrate interacts with the threedimensional surface of the substrate binding pocket of Trypsin. Notice that Asp 189 is located at the end of a hydrophilic pocket (notice the blue and red). Binding of the substrate in this hydrophilic pocket and Asp 189 are essential for good substrate-enzyme interactions. This is the key portion that determines substrate specificity for Trypsin. 90

5 7) Based on your knowledge of the substrate specificity pocket of Trypsin, what other amino acid sidechains would interact well in the pocket? Section 2: HIV Protease and its Substrates Open up the folder titled 2_HIV_Substrate, then double-click on the PyMol file labeled HIV_Protease_Substrate.pml. This will automatically open up the PyMol program, and load up the coordinates for the DNA structure. As you ll recall from David s lectures, HIV Protease is a key protein essential for the post-translational processing of HIV polyproteins into several separate proteins: matrix, capsid, nucleocapsid, protease, reverse transcriptase, and integrase. Like Trypsin, HIV Protease specifically cleaves particular peptide bonds in the polyprotein. This specificity is important in ensuring that the correct proteins are formed. If cleavage was nonspecific, then many different proteins would be produced with different N- and C-terminal amino acids, which could alter their structures and functions significantly. The first image you will see is of the two subunits that form HIV Protease in stick form. If you rotate the structure, you ll notice a hole between the two subunits. This hole is the active site of HIV Protease and is formed by the interaction of the two Protease subunits with one another. Turn off Sticks_A and Sticks_B, and turn on Cartoon_A and Cartoon_B. The active site is less obvious in the cartoon form, however it is still present. In this image, the structures of the two individual subunits is much more apparent. HIV Protease is formed from two identical HIV protease subunits. Thus if we were to look at the primary sequence of the blue subunit, it would be identical to that of the green subunit. More importantly, if these two subunits did not interact with one another, HIV Protease function would be severely impaired as we will see later. 8) In what orientations do the two subunits interact with one another? Click on Substrate The substrate sits in a pocket formed by the junction of the two HIV Protease subunits. Interactions between sidechains of both subunits and the substrate 91

6 are key for defining substrate specificity. Neither subunit alone is sufficient to specifically bind solely the proper substrate. Click on Asp_25. Remember that catalysts are not consumed in the chemical reaction they catalyze. In the case of HIV Protease, this is abundantly clear as Protease helps coordinate a water molecule to cleave the polyprotein backbone. Two of the key residues for this are Asp 25 and Asp 25. The prime is used to notate that the residues are on separate, but identical subunits. Asp 25 and 25 are able to enhance the hydrolysis of the HIV polyprotein by first making water a better nucleophile (positive charge loving) through the deprotonation of water, while simultaneously making a specific carbon atom in the polyprotein a better electrophile (electron loving) by protonating a carbonyl oxygen. For more detail see David s lecture notes. 9) Draw the base catalysis and acid catalysis reaction performed by HIV Protease (hint, see David s lectures). Now if you hadn t noticed, you ll see that two Asn residues are shown rather than Asp residues. This was a key change made for the purposes of crystallizing the enzyme-substrate complex. If the normal protein had been used, it would have cleaved the substrate. This mutant form of HIV Protease is still able to bind the substrate, however it is unable to catalyze the hydrolysis reaction. The enzymesubstrate complex can thus be crystallized and the structure could be solved. 10) Why is the mutant form of HIV Protease shown unable to hydrolyze the substrate? Turn on Ile_50 and Water. The the attacking water molecule is positioned by coordinating it in a precise position with the backbone nitrogens of two Ile residues. This places the water in an ideal position to be acted upon by Asp25, and for the water to simultaneously attack the substrate backbone. Click off Water, Cartoon_A, and Cartoon_B. Turn on Pocket_Residues. As mentioned earlier, the binding pocket is key for ensuring that the enzyme acts upon the proper substrates. In this model the sidechains of HIV Protease that help form the substrate-binding pocket are shown. Notice that the binding pocket is made up of residues from both subunits. 92

7 11) Do you notice anything particularly interesting about the order of the amino acids lining the binding pocket from both subunits (hint, see question 8)? Turn on Binding_Pocket. Now when we look at the surface of the binding pocket, we can see the interaction of the substrate more clearly. Notice the nice fit of the substrate, the interactions that form as well as the multiple openings in the pocket. This is a key feature as HIV Protease acts upon several different protein sequences and is thus able to cleave several different HIV polyproteins with different sequences into the final protein products. 12) Based on the surface representation of the binding pocket, is HIV Protease as selective or less selective of its substrate in comparison with Trypsin? In the next two sections we will examine two different protease inhibitors. 93

8 Section 3: HIV Protease Inhibitors Indinavir Open up the folder titled 3_HIV_Indinavir, then double-click on the PyMol file labeled HIV_Protease_Indinavir.pml. This will automatically open up the PyMol program, and load up the coordinates for Hemoglobin. If HIV Polyproteins cannot be cleaved into individual proteins, the HIV life cycle will be disrupted. Thus a key class of drugs used to inhibit HIV replication are protease inhibitors. These molecules have been specially designed to bind in the active site of HIV Protease and thus prevent Protease from acting upon it s normal substrate. The first model you will see is of HIV Protease in cartoon form. Turn on Indinavir. The Protease inhibitor Indinavir is now shown in stick form. Recall that it is positioned in the active site formed by the interaction of two protease subunits. Indinavir is referred to as a competitive inhibitor meaning it competes with the normal substrate for binding. Turn on Asp_25, Ile_50, and Water Notice that Indinavir is coordinated in the same position as the normal HIV Protease substrate and interacts with the key enzymatic residues, Asp25 and Ile50, in the same way. Also notice that in this model Asp_25 is really represented by Asp residues rather than Asn residues. If you zoom in on Indinavir, you will notice that the backbone chain is not the same as a normal peptide backbone. This change prevents it from being cleaved by the protease. 13) Why could a crystal structure be made of Indinavir with normal HIV Protease? Turn on Binding_Pocket. When we look at the binding pocket we see that Indinavir fits well and makes many positive interactions with the HIV Protease binding pocket sidechains. Thus it is clear that it is able to form many of the same positive interactions as the normal substrate, but is uncleavable. 14) For Indinavir to be an effective inhibitor, why is it important that it cannot be cleaved? 94

9 Section 4: HIV Protease Inhibitors Dupont Open up the folder titled 4_HIV_DuPont, then double-click on the PyMol file labeled HIV_Protease_DuPont.pml. This will automatically open up the PyMol program, and load up the coordinates for Top7. As you saw in the previous section, Protease Inhibitors can be designed that bind in similar fashions as natural substrates. However in this section we will look at a newer Protease Inhibitor with enhanced binding affinity and study why it binds better. Turn on Dupont. It is clear that the DuPont inhibitor binds in the active site of HIV Protease just as Indinavir does. However if you look at the structure more closely, you ll notice a key difference in the molecule. At the center of the molecule is a 7- member ring. Ring structures are inherently more rigid than linear structures. 15) What thermodynamic effect will the presence of a ring in the inhibtor have upon its binding to HIV Protease? Turn off Cartoon_A and Cartoon_B. Turn on Asp_25 and Pocket_Residues. In this model we still see that the DuPont inhibitor is well coordinated with the pocket residues and the key Asp25 catalytic residues. No clear explanation can yet be seen for the increased binding affinity of this molecule over Indinavir. Turn off Asp_25 and Pocket_Residues. Turn on Binding_Pocket. The same nice fit is seen when the binding pocket surface representation is shown. Turn off Binding_Pocket, turn on Ile_50, Asp_25 and Water. The carbonyl oxygen connected to the 7-member ring in the middle of the molecule is coordinated with Ile50 and Ile50. Normally Ile50 and Ile50 coordinate a water molecule to attack the substrate. By specifically placing a carbonyl oxygen on the inhibitor to coordinate with Ile50 and Ile50, one extra water molecule is liberated from interacting with the protease. 16) What will be the thermodynamic effect of liberating the water molecule normally used to attack the substrate upon HIV Protease binding? 95

10 Section 5: HMG-CoA Reductase and HMG-CoA Open up the folder titled 5_HMG_Substrate, then double-click on the PyMol file labeled HMG_Substrate.pml. This will automatically open up the PyMol program, and load up the coordinates for bulk water. For more information about HMG-CoA Reductase, see the appendix attached to Lab 5 and more information is included with Lab 7. Cholesterol is synthesized in the cell through a series of chemical reactions, each using a different enzyme. One of the key steps in cholesterol biosynthesis is the conversion of HMG-CoA to Mevalonate using HMG-CoA Reductase. This is considered the first committed step as once a molecule of HMG-CoA is converted to mevalonate, it is committed to the cholesterol biosynthetic pathway. 17) Uncatalyzed, the reaction converting HMG-CoA to Mevalonate is extremely slow. HMG-CoA Reductase accelerates this reaction markedly. On a reaction energy diagram, draw the graph that would be expected for the spontaneous conversion of HMG-CoA to Mevalonate in the presence and absence of HMG- CoA Reductase. Include in your drawing ΔG and ΔG rxn The first model you will see is of a complete HMG-CoA Reductase molecule. 18) How many individual polypeptide chains form a single HMG-CoA Reductase protein? Turn off Full_Tetramer and turn on Interface_A and Interface_B. While the full structure is interesting, it is a bit cumbersome to use. In this model you can see the interlinking fit between two of the HMG-CoA Reductase subunits. Turn off Interface_A and Interface_B. Turn on Cartoon_A, Cartoon_B, Coenzyme_A, and HMG. This model shows the substrate interacting with the enzyme. Normally HMG and CoA are connected as a single molecule when binding to the enzyme. However in this case they are shown already cleaved and separated. Turn on NADP. For each molecule of HMG-CoA, two molecules of NADPH are used to reduce HMG-CoA to Mevalonate and CoenzymeA. In the process, these two molecules of NADPH are oxidized into NADP +. 96

11 19) In one of Dan s lectures, he spoke of a specific oxidation and reduction reaction with regards to protein structure. What other example of oxidation and reduction have we seen in the course? (hint, think perms). Turn off Cartoon_A and Cartoon_B. Turn on Binding_Pocket. Notice how the HMG interacts well with the binding pocket, but the CoA doesn t interact much. Thus much of the binding of HMG-CoA to HMG-CoA Reductase is driven by the HMG portion, and not the CoA portion of the molecule. 97

12 Section 6: HMG-CoA Reductase and Fluvastatin Open up the folder titled 6_HMG_Drug, then double-click on the PyMol file labeled HMG_Drug.pml. This will automatically open up the PyMol program, and load up the coordinates for bulk water. As you ve already learned, Fluvastatin inhibits the function of HMG-CoA Reductase, and thus decreases the biosynthesis of cholesterol. In this model, only two of the four subunits of HMG-CoA Reductase are shown. Turn on Fluvastatin You ll notice that Fluvastatin has a moiety that is very similar to HMG. This portion is key for its ability to bind in the active site of HMG-CoA Reductase. Also observe that similar to HIV Protease Inhibitors, Fluvastatin cannot be reduced to form two separate molecules. Thus an individual molecule of Fluvastatin can inhibit HMG-CoA Reductase for a long period of time. Turn on Binding_Pocket. Observe the binding of Fluvastatin. As mentioned previously, the HMG moiety of Fluvastatin allows it to bind in the active site of HMG-CoA Reductase. However for the molecule to be useful as an inhibitor, it needs to bind with greater affinity. 20) What effect will the relative affinity of an enzyme for an inhibitor versus its natural substrate have upon the concentration or dose of the inhibitor required to inhibit normal enzyme function? As you ll recall from the appendix of Lab 5, all Statins also possess hydrophobicring structures. These ring structures interact with a hydrophobic patch near the active site of HMG-CoA Reductase, thus increasing binding affinity. The normal substrate, HMG-CoA lacks the hydrophobic patch and thus does not form this interaction. 21) Thermodynamically, how does the presence of the hydrophobic rings increase binding affinity? Is entropy or enthalpy more affected? 98