Quaternary Structure & Conformational Diseases PDF

No. 3 Quaternary structure – role in regulation of biological activity of proteins The present lecture material was mainly created by Prof. Ana Maneva, DSc, Assoc. Prof. Anelia Bivolarska, Ph.D., M.D., and Prof. Tatyana Vlaykova, Ph.D., from the Department of Medi...

No. 3 Quaternary structure – role in regulation of biological activity of proteins The present lecture material was mainly created by Prof. Ana Maneva, DSc, Assoc. Prof. Anelia Bivolarska, Ph.D., M.D., and Prof. Tatyana Vlaykova, Ph.D., from the Department of Medicinal Biochemistry at the Medical University of Plovdiv.  Quaternary structure ✓ The quaternary structure is determined by the spatial arrangement of two or more polypeptide chains (called subunits) forming a common complex. ✓ Each subunit has its own primary, secondary and tertiary structure. ✓ The primary structure of the subunits can be the same or different and, accordingly, the proteins are homo- or hetero-oligomers. ✓ The subunits are linked by non-covalent interactions (hydrogen bonds, hydrophobic and electrostatic interactions). ✓ Some of the subunits perform a catalytic function, while others perform recognition and regulatory functions. ✓ A change in the spatial arrangement of the subunits changes the properties of the molecule. Therefore, proteins with a quaternary structure have an important role in the regulation of intracellular processes.  Quaternary structure Hemoglobin is an example of a quaternary protein. The four subunits as well as the four hemes are represented by a different color. Hemoglobin Proteins with a quaternary structure - Stores Fe in the cells  Mechanisms for maintaining the conformation of proteins in the cell  Denaturation and renaturation ✓ Denaturation is a process in which, under the influence of various chemical and physical agents (high temperature, acids, bases, detergents, radiation), the conformation of the molecule is disturbed, the physicochemical properties change and the biological activity is lost. ✓ It destroys the quaternary (if any), tertiary and secondary structures, but not the primary structure of the protein. ✓ It can be reversible or irreversible. In reversible denaturation, after removal of the denaturing effect, the molecule again assumes a native conformation (renaturation). ✓ In cells, there are mechanisms that maintain the conformation of proteins and assist in the correct folding of the polypeptide chain. A scheme for the denaturation of the ribonuclease enzyme under the influence of urea, destroying the disulfide bridges and the conformation of the enzyme, is shown. The state is reversible under certain conditions and the return to the correct conformation is called renaturation. Folding of newly synthesized polypeptide chains There are special proteins in the cells that facilitate the folding process. These are: 1. protein disulfide isomerases that catalyze displacement of disulfide bonds when they are formed at sites not determined by the primary structure. 2. prolyl cis-trans isomerases. 3. chaperones Protein disulfide isomerase (PDI) PDI contains an active site with two reduced cysteine ​sulfhydryl groups (– SH). An ionized form of one of these groups (–S−) reacts with a disulfide bridge (S – S) on proteins to form a disulfide-linked intermediate enzyme- substrate complex between PDI and the protein. In this complex, a free ionized -S− group is formed on the protein, which, in turn, can react with another disulfide bridge in the protein, forming a new disulfide bridge and another free ionized -S− group. In this way, the disulfide bridges on a protein can rearrange each other until the most stable conformation is achieved and the enzyme is released.  Peptidyl prolyl cis-trans isomerases (PPIase) (cyclophilins) Some proteins contain a cis X-Pro bound in the native structure, so molecules with a trans X-Pro bond must convert to the cis isomeric form to complete the protein conformation. PPIase catalyzes the cis/trans conversion, thereby increasing the efficiency of creating the native conformation by about 300-fold. Inhibition of cyclophilins by cyclosporins and FK506 results in immunosuppression and is useful in organ transplantation to suppress transplant rejection. Families of PPIase proteins. (A) PPI activity: cis-trans isomerization of X-Pro. (B) Crystal structures of human PPIases from the four different families: cyclophilins (CyP), FK506-binding proteins (FKBP), parvulins (Par), and protein phosphatase two A phosphatase activator (PTPA). Cyclophilin A is shown in dark blue (PDB: 3K0M). FKBP12 (PDB: 2PPN). PIN1 is indicated in green (PDB 1PIN). PTPA (PDB 2IXM) is indicated in gray. (C) Inhibitors of different PPIase families. https://doi.org/10.3390/pathogens13080644  Chaperones Chaperones (70 kDa) are a family of proteins that, under physiological conditions, prevent misfolding and "unwanted" interactions between amino acid radicals in newly synthesized polypeptide chains. The primary structure of a protein determines its higher structures, but chaperones facilitate reaching the thermodynamically most stable protein conformation. For the correct folding of the nascent polypeptide chain, chaperones bind temporarily to the hydrophobic regions of the nascent chain, protecting them from solvent exposure. To carry out their activity, chaperones require energy in the form of ATP (adenosine triphosphate).  Features of chaperones 1. Chaperones are proteins with a quaternary structure that create protective cavities for many proteins. 2. Chaperones are also called heat-shock or stress proteins because they are extensively synthesized during heat shock, probably to counteract the denaturation of cellular proteins. 3. Chaperones also facilitate the transport of proteins in different cellular compartments. E.g. one chaperone maintains the newly synthesized protein in an unfolded state until it passes through the mitochondrial membrane, and another chaperone in the matrix facilitates protein folding. 4. Chaperones are involved in the signal transduction of steroid hormones that use intracellular receptors. 5. Chaperones are also involved in the removal mechanisms of proteins with incorrect conformation, participating in directing them to proteolytic degradation in the proteasome. Molecular models of chaperones that form protective cavities for proteins Schematic showing mitochondrial transport and creation of a native conformation of a precursor protein Protein enters the mitochondria and undergoes further processing there. In order for it to be properly carried out, two chaperones are involved – Hsp70 and Hsp 60. The heat shock protein Hsp104 assists two other heat shock proteins - Hsp70, Hsp40 to restore the native conformation of aggregated proteins, such as occur in some pathological conditions (e.g. neurodegenerative)  Chaperones are involved in steroid hormone signaling NR = nuclear receptor HSP = heat shock protein HRE = hormone responsive region of DNA When the steroid hormone binds to the receptors, the chaperones (hsp) are released from the complex and the receptor forms a dimer with another identical and activated receptor. The dimer is then free to enter the nucleus, where it binds to a specific DNA sequence.  Relationship between protein structure and biological function  The function of a protein depends on its conformation. If it is broken, the protein denatures and loses its activity. Examples: Denatured enzymes lose their catalytic ability. Denatured antibodies can no longer bind antigen. A mutation in the gene encoding the protein is a common cause of a change in the tertiary structure. The mutated protein cannot fulfill its exact purpose in the cell and/or be degraded. For example, in Osteogenesis imperfecta, it is caused by an inability of mutated type I collagen to assemble properly. Examples of defects in receptors: 1. Diabetes insipidus is caused by failed folding of a mutated variant of the V2 gene for the vasopressin (ADH) receptor. Familial forms of diabetes insipidus: Clinical and molecular characteristics Jul 2011 · Nature Reviews Endocrinology Scheme of vasopressin V2 receptor with mutations associated with severe and partial forms of Diabetes insipidus. 2. Familial hypercholesterolemia is caused by a mutation in the low-density lipoprotein receptor (LDL-R) gene. Examples of diseases due to disordered protein conformation: The mutant proteins aggregate and form non-functional, insoluble aggregates. 1. Alzheimer's disease - insoluble aggregates called amyloid form in the brain. George Mason University Department of Bioengenering, Komine H et al., 2015  2. Prion disease ("mad cow" desease, in humans known as Creutzfeldt-Jakob disease) The normal protein has Protease many α-helical regions and Amino acids is soluble. In the mutant variant, the α-helix is ​converted to a β-helix and Protease the protein becomes Amino acids insoluble. Unlike the normal protein, the mutant is resistant to the action of proteases. Some of the neurons degenerate because insoluble amyloid fibrils containing pathological prion proteins are deposited in them. α-helices are marked in green, and the β-helices – in blue on the diagram  Huntington’s disease – a conformational disease A genetic mutation of the HTT gene causes Huntington’s disease. The HTT gene makes a protein called huntingtin. This protein helps your nerve cells (neurons) function. If you have Huntington’s disease, your DNA doesn’t have all the information needed to make the huntingtin protein. As a result, these proteins grow in an abnormal shape and destroy (instead of help) your neurons. Your neurons die because of this genetic mutation. The destruction of nerve cells happens in the basal ganglia or the region of your brain that regulates your body’s movements. It also affects the brain cortex (surface of your brain) that regulates your thinking, decision-making and memory. https://www.fcneurology.net/what-is-huntingtons-disease/  Expression of point mutations - Sickle cell anemia The replacement of polar glutamate in HbA with hydrophobic valine in HbS changes the conformation and physicochemical properties of the protein molecule, facilitates the polymerization and precipitation of HbS, and leads to hemolysis of erythrocytes.  Cystic Fibrosis (CF, Cystic Fibrosis) CF is caused by a mutation in the CF transmembrane conductance regulator (CFTR) gene. The CFTR protein produced by this gene regulates the movement of chloride and sodium ions across epithelial cell membranes. When mutations occur in one or both copies of the gene, ion transport is defective and leads to a buildup of thick mucus throughout the body, causing respiratory failure along with many other systemic obstructions and abnormalities. A combination of reduced mucociliary clearance and altered ion transport allow bacterial colonization of the airways, most commonly Pseudomonas, Haemophilus influenza, and Staphylococcus aureus. These pathogens trigger a massive inflammatory response. Eventually, chronic infection and this repeated inflammatory response can lead to airway destruction. CFTR is thought to regulate other ion channels, for example ENaC (for sodium transport). The ΔF508 mutation, the deletion of phenylalanine at position 508 of the polypeptide, is present in 70% of CF chromosomes, although more than 1000 other mutations have been documented in the CF gene. The ΔF508 mutation leads to abnormal synthesis and premature degradation of CFTR protein and as a result there is a deficiency/absence of CFTR in the apical membrane of bronchial epithelial cells. This causes decreased permeability for chloride ions across the epithelium. CF patients exhibit pulmonary disease consistent with a failure of innate airway defense mechanisms. Abnormal ion transport leads to dehydration of airway mucus. Drug therapy targets the ion transport defects with the intention of rehydrating the airway surfaces. DOI 10.1007/s10495-013-0874-y DOI 10.1097/01.JAA.0000515540.36581.92 Cystic Fibrosis (CF, Cystic Fibrosis) https://www.immunology.org/public- information/bitesized- immunology/pathogens-disease/microbia infection-cystic-fibrosis DOI: 10.5772/intechopen.97537 https://web.stanford.edu/class/psych1 21/humangenome-CF.htm  2,3-Bisphosphoglycerate is a regulator of oxygen affinity for hemoglobin 2,3-bisphosphoglycerate HbA binds the negatively charged 2,3-bisphosphoglycerate in the gap between the four subunits, and this lowers the affinity of Hb for O2. This facilitates the delivery of oxygen to the tissues. Three positively charged groups of each β- subunit, one of which is His21, are involved in binding. Fetal HbF contains γ chains instead of β chains. In the γ chains, His21 is replaced by Ser. Therefore, HbF has a lower affinity for 2,3-phosphoglycerate and the latter does not inhibit the binding of O2 to HbF. This allows HbF to readily bind O2 released from maternal HbA.  Defects in post-translational processing of proteins Involvement of ascorbic acid (vitamin C) in the hydroxylation of proline and lysine in the process of collagen maturation. Scurvy develops when there is a dietary deficiency of vitamin C (ascorbic acid), which is a necessary cofactor for the enzymes prolylhydroxylase and lysylhydroxylase. As the name suggests, these enzymes catalyze the covalent modification of proline and lysine - hydroxylating them to hydroxyproline and hydroxylysine, respectively. These enzymes do not hydroxylate free amino acids, but only those already included in a polypeptide chain. Lysine, hydroxylysine, proline and hydroxyproline are involved in the formation of stable cross-covalent bonds, enhancing fiber strength. Hydroxylation Prolyl-hydroxylase Lysyl-hydroxylase Glycosylation Assembly of 3 pro-α chain Procollagen triple helix formation DOI:10.3390/nu7042324 Hydroxylation of lysine and proline  Non-enzymatic glycation of hemoglobin In diabetes, the level of blood glucose increases, which can bind to serum proteins and hemoglobin. The half-life of erythrocytes is approx. 60 days, so HbA1c gives information about the average concentration of glucose during the previous 1.5-2 months. The level of the so-called glycated hemoglobin (HbA1c) is high in diabetics and is used as an indicator of metabolic control in diabetes.