Proteins: Sequence --> Structure --> Function Anfinsen Experiment: Denature ribonuclease (RNase) Remove denaturant Assay for RNase activity -- does the protein regain its 3-D structure and its enzymatic activity?

Molecular chaperones - Anfinsen cages for folding proteins Xu et al., 1997, Nature 388: 741 GroEL-GroES-(ADP)7 complex

Inverse Protein Folding Problem Given a structure (or a functionality), identify an amino acid sequence whose fold will be that structure (exhibit that functionality). Can we make designer proteins with desired functions?

Central dogma I -- Replication DNA -> RNA -> protein But for retroviruses, DNA can also be made by reverse transcription of RNA. To understand the lifecycle of a retrovirus, we need to know more about how DNA is replicated and transcribed, and how RNA is translated into protein.

Vocabulary Replication -- copying DNA before cell division Transcription -- making an RNA copy (messenger RNA or mRNA) of DNA Translation -- making a protein from the mRNA DNA ---------> DNA DNA ---------> RNA ----------> Protein RNA ---------> DNA replication transcription translation reverse transcription

Polymerase enzymes DNA ---------> DNA DNA ---------> RNA RNA ---------> DNA replication (DNA polymerase plus other proteins) transcription (RNA polymerase plus other proteins) reverse transcription (HIV reverse transcriptase) HIV reverse transcriptase (RT) is a RNA-dependent DNA polymerase with many similarities to host cell DNA polymerase, therefore we will discuss RT in this lecture.

Genes are the functional units of heredity The genetic information carried in DNA must be duplicated before a cell can produce two genetically identical daughter cells. The genetic information carried in DNA is in the form of genes: a gene is a segment of DNA containing the instructions for making a protein or set of closely-related proteins. Cells contain elaborate machines to accurately copy or replicate their DNA and to repair it when it is damaged (e.g., by chemicals or environmental radiation).

The structure of DNA explains how genetic information is copied Each strand of the DNA double helix is complementary to its partner strand, so each can act as a template for synthesis of a new complementary strand (“semi-conservative” replication). Base-pairing allows a simple way for cells to pass on their genes to descendents.

What you need to remember about DNA structure to understand replication

Clicker question DNA can be used to: Code for proteins Make origami pictures Build smaller, faster computers All of the above

Clicker question DNA can be used to: Code for proteins Make origami pictures Build smaller, faster computers All of the above B & C will be discussed in the next lecture

DNA replication Begins at an A-T rich replication origin. Initiator proteins bind and separate the two DNA strands. A protein machine containing DNA polymerase is assembled. DNA polymerase synthesizes new DNA using one old strand as a template. Replication forks move bidirectionally from origins of replication.

Clicker question Why are origins of replication AT-rich? DNA polymerase binds AT basepairs with high affinity. AT-rich regions are easier to copy. The free energy required to separate AT-rich regions is lower than for GC-rich regions. Origins always start with the first letter of the alphabet, hence they must start at an “A”. DNA is almost entirely AT-rich, so there’s a higher probability that replication will begin at an AT-rich than a GC-rich region.

DNA always synthesized in 5’ to 3’ direction A new deoxyribonucleotide is always added to the 3’ OH end of the new strand. One new DNA strand at the replication fork is made on a template that runs 3’ to 5’; the other strand is made on a template that runs 5’ to 3’. How can the top strand be made in the direction of the replication fork?

Clicker question The top strand is synthesized backwards. DNA must be synthesized in the 5’ to 3’ direction. How is it possible to make two new strands, both going in the direction of the replication fork? The top strand is synthesized backwards. The top strand makes a parallel double helix with the DNA strand from the parental helix. The top strand is synthesized in short 5’ - 3’ pieces, then stitched together. Individual nucleotides basepair with the top strand in the parental DNA helix, then covalently bond with each other to form a new strand.

One DNA strand is synthesized continuously; the other in synthesized discontinuously and then stitched together 5’ 3’ 3’ 5’ Leading strand: DNA strand that is synthesized continuously in the direction of replication fork. Lagging strand: DNA strand that is synthesized discontinuously (Okazaki fragments), then stitched together.

DNA polymerase video

DNA polymerase is part of a protein machine that replicates DNA -- Sliding clamp is another component

DNA replication (DNA interactive video)

DNA polymerase corrects its mistakes to avoid mutations DNA polymerase error rate: 1 mistake per 107 nucleotides. Proofreading activity of DNA polymerase: if it adds an incorrect nucleotide, it cleaves the phophodiester bond (acts as a 3’ - 5’ exonuclease as well as a polymerase), then tries again. DNA replication error rate including mismatch repair: 1 mistake per 109 nucleotides.

“P” is the catalytic site for the polymerization activity. DNA polymerase structure shows separate sites for DNA synthesis and for editing “E” is the catalytic site for the error-correcting exonuclease activity. “P” is the catalytic site for the polymerization activity.

DNA Mismatch Repair System serves as a backup to prevent copying mistakes Fidelity of DNA replication increases 100-fold when add in mismatch repair system: 1 mistake in 107 nucleotides for DNA polymerase alone compared with 1 mistake in 109 nucleotides.

What is the problem with mutations? Genetic changes are what drive evolution -- they allow organisms to adapt to changing conditions and colonize new habitats. BUT … from the perspective of a single organism (e.g., you or me), a permanent genetic change (mutation) can have profoundly negative consequences: Sickle cell anemia, an inherited disease, is caused by a change in one nucleotide leading to a single amino acid change in hemoglobin. Cancers are caused by a gradual accumulation of random mutations in DNA of somatic* cells. From the perspective of a virus, mutations are great because they allow emergence of rare variants that have increased fitness and/or can evade the host immune system more effectively. *Somatic cells are all the cells in an organism other than germ cells (the reproductive cells).

HIV reverse transcriptase is a low-fidelity DNA polymerase with no proof-reading activity *p66 subunit of HIV RT Klenow fragment of DNA pol *Note on nomenclature: proteins are often named pX, where X is the molecular weight in kilodaltons. All polymerases have a common architectural framework consisting of three canonical subdomains termed the fingers, palm, and thumb subdomains. HIV RT is a DNA polymerase that can use DNA or RNA as a template. Kohlstaedt et al., 1992, Science 256, 1783-1790

Reverse transcription is critical for the lifecycle of a retrovirus 06_39_retrovirus.jpg

Structural studies of HIV reverse transcriptase RT is a heterodimer containing two subunits: p66 and p51 (p51 is derived from p66 by proteolytic cleavage). Note on nomenclature: proteins are often named pX, where X is the molecular weight in kilodaltons. Polymerase domains p66 and p51 each contain fingers, palm and thumb subdomains. Cleft between subdomains in p61 polymerase domain binds template-primer. Cleft is closed in p51 subunit. p51 contains only a polymerase domain. p66 includes a C-terminal RNase H domain, which degrades the RNA strand of an RNA/DNA hybrid. Tantillo et al., 1994, J. Mol. Biol. 243: 369-387

HIV reverse transcriptase -- Two enzymes in one After building a DNA strand from the RNA template using polymerase activity, the RNase activity destroys the RNA strand, then a second DNA strand is constructed from the first by the polymerase. http://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb33_2.html

HIV reverse transcriptase has no proof-reading activity In vitro assays suggest error rates of ~1/1700 nucleotides (compare with 1/10,000,000 for DNA polymerase). Base substitution, addition, deletion errors. Some template positions are mutational hotspots with error rates of 1/70 nucleotides. The exceptional diversity of the HIV-1 genome results from error-prone reverse transcription. Most of the mutant viruses are inferior or not viable, but some are better than the parental virus at escaping from the immune system or from anti-viral drugs. Natural selection at work! Roberts et al., 1988, Science 242: 1171-1173.

What should you target to make an anti-viral drug? Clicker question What should you target to make an anti-viral drug? A) An activity that is critical for viral function B) An activity that is virally-encoded C) An activity that is not similar to host activities D) All of the above

What should you target to make an anti-viral drug? Clicker question What should you target to make an anti-viral drug? A) An activity that is critical for viral function B) An activity that is virally-encoded C) An activity that is not similar to host activities D) All of the above Logical candidate for an anti-retroviral drug: Reverse transcriptase

Reverse transcriptase inhibitors RT inhibitors prevent synthesis of double stranded viral DNA, thus prevent HIV from multiplying Nucleoside analog RT inhibitors (NARTIs or NRTIs) Competitive substrate inhibitors Lacks 3’ OH so causes chain termination Example: AZT, the first anti-HIV drug Conversion to nucleotide by phosphorylation in body can cause toxicity Nucleotide analog RT inhibitors (NtARTIs or NtRTIs) Competitive substrate inhibitors Lacks 3’ OH so causes chain termination Doesn’t need to be converted by body, so less toxic Non-nucleoside RT inhibitors (NNRTIs) Non-competitive substrate inhibitors Not incorporated into viral DNA Inhibit polymerase through conformational changes in active site

Crystal structure of HIV RT bound to Nevirapine, a NNRTI that binds near, but not in, the polymerase active site PDB code 1jlb The PDB contains >100 RT/inhibitor complexes. http://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb33_2.html

FDA-approved reverse transcriptase inhibitors http://www.hivandhepatitis.com/hiv_and_aids/hiv_treat.html

What should you target to make an anti-viral drug? An activity that is critical for viral function An activity that is virally-encoded An activity that is not similar to host activities but RT is a polymerase similar to host polymerases… Fortunately NRTIs and NtRTIs bind RT more tightly (higher affinity) than they bind DNA polymerase. Also if they did get incorporated by DNA polymerase, NRTIs would be removed from host cell DNA during DNA repair. HOWEVER -- mitochondrial DNA is replicated by polymerase gamma, which binds NRTIs, so mitochondrial DNA can be damaged, resulting in cell death due to low energy production. Different NRTIs affect mitochondria of different types of cells, so have different side effects.

Side effects of some reverse transcriptase inhibitors include: Headaches, high blood pressure, nausea, vomiting, fatigue (can disappear with time) Less frequent, but more serious side effects include anemia (shortage of red blood cells), myopathy (muscle pain and weakness), neutropenia (low number of neutrophils) Note: RT inhibitors are usually used in combination with other types of anti-retroviral drugs (protease or fusion inhibitors).