Take-Home Messages - Architecture of Biopolymers PDF

i will upload take home messages of all presentations of my subject ''Architecture of Biopolymers''. Make a document with all important points from each topic that i need to study and remember a day before exams. make a downloadable document and make the notes aesthetic and well structured. 1. Prin...

i will upload take home messages of all presentations of my subject ''Architecture of Biopolymers''. Make a document with all important points from each topic that i need to study and remember a day before exams. make a downloadable document and make the notes aesthetic and well structured. 1. Principles of protein structure 1. Stability and flexibility of peptide chains are influenced by resonance, coplanar rigid bonds and rotable N-Cα and Cα-C bonds. 2. Mainly noncovalent interactions (especially van der Waals interactions, hydrogen bonds, salt bridges), but also covalent bonds (especially peptide bonds, disulfide bridges) stabilize folded proteins. 3. While polar and charged side chains are often found on the protein surface where they can bind water, nonpolar side chains build the hydrophobic core of the protein. 4. Loops of amino acids in between the secondary structural elements often serve as sites for protein recognition, ligand binding and membrane interaction. 5. Proteins are only marginally stable (essential for flexibility and function). 6. The five classes of domain folds are alpha domains, beta domains, alpha/beta domains (Beta- alpha-beta-alpha motif), alpha+beta domains (separate beta sheet and alpha helix motifs) and cross-linked domains (with disulfide bridges or metal ion). 7. Protein motions like fluctuations, collective motions of bonded and non-bonded neighbouring groups of atoms and ligand-induced conformational changes influence the flexibility of proteins 2. Experimental methods for structure determination The steps of an X-ray structure determination include:  protein crystallization  irradiation ofthecrystal withX-rays  collection of a diffractionpatternresulting fromscatteredX-rays  computationofelectrondensitymaps  constructionofanatomicprotein model  iterative model refinement Take-home-messages I: General aspects of X-ray crystallography Parameters that critically affect crystal formation:  pH  temperature  proteinconcentration  proteinpurity  typeofthesolventandprecipitant  presenceofionsorligands Take-home-messages II: Interpretation of X-ray structures Measures of quality include:  R-factor(measure of the agreement between the crystallographic model and the experimental X-ray diffraction data; 0.0 for exact agreement; 0.59 for total disagreement)  Rfree(global measure of model-to-data agreement calculated only for ~1000 randomly selected reflections that were never used for model refinement)  Root-mean-square deviations (RMSDs) from stereochemical standards (indicates how much the model deviates from geometrical parameters that are considered typical, based on previous experience)  sidechain outliers (percentage of residues with unusual sidechain conformations)  steric clashes (number of pairs of atoms which are unusually close together)  ramachandranplot outliers (residues with unusual φ/ψ backbone torsion angles) Resolution: Smallest distance of atoms to appear as separate maxima in the electron density plot o ~ 6 Å –shapeof the macromolecule o < 3 Å –the polypeptide chaincan be traced o < 1.5 Å –positions of individual non-hydrogen atomsstart to become resolved o < 1 Å –direct location of hydrogen atoms in the electron-density map o 0.77 Å –physical limit, when using copper KαX-ray radiation o < 0.77 Å –only very few protein structures available The appearance of electron density as a function of the resolution of the experimental data 3. Prediction of protein structures Homology modelling includes the following steps:  Framework construction  Building non-conserved loops  Completing the backbone  Adding sidechains  Energy minimisation Take-Home-Messages I Basic steps of Homology Modelling A good template for Homology Modelling should...  cover the entire length of the target sequence  produce a high GMQE-score  exhibit a high sequence identity to the target sequence  be available at a high structural resolution  reflect the correct oligomerization state for the target sequence  contain all physiologically relevant ligands Take-Home-Messages II Fold recognition (‘threading’) methods  do not require sequence homology  require at least one homologue of known fold for successful predictions  score models with potentials of mean force  frequently yield only ‘low resolution’ models Steps and output of AlphaFold predictions include  analysis of sequence features and covariance patterns  application of a deep neural network for predicting the distances between residues  a gradient descent procedure to refine the protein structure  the position-specific pLDDT score indicating the local confidence of the model:very high (pLDDT > 90); confident (90 > pLDDT > 70); low (70 > pLDDT > 50); very low (pLDDT < 50)  a ‘predicted alignment error’ indicating the reliability of intramolecular distances Recent extensions of AlphaFold 2 include  structure prediction for multimeric proteins (AlphaFold 3)  prediction of multiple conformational states for the same protein (biased AF)  identification of disordered protein regions (AF-disorder)  prediction of the effect of mutations on protein function (AlphaMissense)  addition of ligands or ions to modelled protein structures (AlphaFill) 4, Eukaryotic DNA binding proteins Take-home messages 80% of DNA-binding domains are from one of the families: Helix-Turn-Helix, Helix-Loop-Helix, zinc-containing motifs, and leucine zippers. TBP is a saddle-shaped molecule that bends and unwinds the TATA box ○ Binds the minor groove of DNA and the interac on is hydrophobic, unlike other TFs ○ Forms salt bridges between Arg, Lysine and phosphate backbone of DNA for stabilisa on ○ DNA binding by TBP depends on the presence of TA bps, that allows bending of DNA Homeobox proteins contain a helix-turn-helix motif and interact as monomers with both the major and minor grooves p53 consists of a transac va on domain, a DNA-binding domain, and an oligomerization domain The DNA-binding domain of p53 consists of an antiparallel β-barrel (with 9 β-strands) with an immunoglobulin fold Most carcinogenic muta ons in p53 occur in the DNA binding regions 215. Prokaryotic DNA- binding Proteins Take home messages Medizinische Fakultät 11. November 2024 24 DNA Recognition in Procaryotes by Helix-Turn-Helix Motifs Thehelix-turn-helixmotifincludesarecognitionα-helixthatbindsinthemajorgrooveofB- DNAandastabilizationα-helix,connectedbyashortturn. Subunitinteractionsensurethecorrectdistanceof34Åandorientationbetweenthedimer'srecognitionα- helices,increasingaffinitybetweenproteinandoperatorDNA Sequence-specificinteractionbetweentherecognitionα- helixandDNAfacilitateidentificationofthepalindromicoperatorregion. Hydrogenbondsbetweensugarphosphatebackboneandtheprotein,alongwithDNAdistortion,allowforcl oseinteractionbetweenoperatorregionsandDNA- bindingproteins,influencingdifferentialbindingaffinitiesforCroandrepressor https://dccdn.de/www.doccheck.com/data/mp/re/nq/v6/2t/bg/hom_odom_ne_md.jpg Helix-turn-helix motif, bound to DNA Medizinische Fakultät 25 Tetracycline induces expression of tetA by binding to TetR and causing conformational change Structure of TetR Homodimer; 10 α-helices per polypeptide chain Two separate domains: DNA-binding domain (with HTH-motif) and regulatory domain ([MgTc]+ binding pocket) TetR-tetO interactions are sequence specific and of high structural complementary Interactions due to specific hydrogen bonds between TetR and tetO; no H2O in DNA-protein interafce No overall DNA bending (kink at base pair 2 is compensated) Take-home messages Medizinische Fakultät 11. November 2024 26 Induction by tetracycline and Mg2+ leads to the dissociation of the TetR-tetO complex Conformational changes thoughout the entire protein α4 swings in a pendulum-like motion → distance between recogni on helices (α3 and α3´) is widened by ca. 3 Å → Disrup on of the DNA-binding domain-half operator contacts → dissocia on of TetR-tetO complex Shared features of TetR and LacI repressors Dimers (LacI dimer of dimers) separate domains for DNA- and inducer-binding: HTH-motif interacts with symmetrical DNA sequence, rigid region in ligand-binding domain Distinct LacI features Overall DNA-bending (45°) upon repression Induction by β-1,6-allolactose: unfolding of hinge helices → free HTH-motif Lower affinity to operator DNA compared to TetR Take-home messages Serine proteinases Medizinische Fakultät 15. November 2024 23 Serine proteinases cleave peptide bonds in a two-step mechanism: First the peptide bond undergoes a nucleophilic attack by the Serine forming a tetrahedral transition state from which one part of the peptide (N-terminal) is released, then theenzyme recovers to its original state by deacetylation. In generalthey have four characteristic features: CatalytictriadconsistingofSer,HisandAspresidues Oxyanionholeforstabilizationofthetransitionstate Main-chainsubstratebindingfornon-specificbinding Specificitypocketthatmakesupthepreferenceforcertainsubstrates Serineproteinasesbelongtodifferentfamilies,viz;chymotrypsinlike,dipeptidyldeptidaseIV- like,subtilisin-like,serine-carboxylprotease,serine-lysineproteases,andrhomboids. Chymotrypsin-likeSerineproteinaseshaveatwo-domainbeta- barrelstructurewithacatalytictriadofserine,histidine,andaspartate,whicharelocatedinloopsofdomain2 anddomain1,respectively. Subtilisin-likeSerineproteinasesarestructurallyunrelatedtothechymotrypsin- clanofserineproteinasesbutusesthesametypeofcatalytictriadintheactivesite.Thismakesitaclassicexam pleofconvergentevolution.Eg:SubtilaseCytotoxin(SubAB) Take Home Messages HIV-1 Protease TAKE-HOME 1 HIV1-Protease cleaves the viral polyprotein and releases active structural proteins needed for virus replication. HIV1-Protease is a symmetric homodimer consisting of 99 aminoacids per monomer. Two Asp-25 residues from both monomers form the central active site. Flexible „Flaps“ can bind the substrate using hydrogen bonds while causing conformational changes. TAKE-HOME 2 HIV1-Protease cleaves the P1-P1‘ peptide bond by means of hydrolisation. Protease-inhibitors such as Darunavir have been effectively used as treatment. Mimicking the hydrogen bonds between the substrate, ac ve site, and flaps was exploited in drug design to target mutated virus strains. tyrosine kinases...TAKE HOME MESSAGES 15 the evolutionarily similar structures of c-Src and c-Abl have been adapted for different regulatory mechanisms c-Src achieves its autoinhibition by binding of the SH2 domain to a phosphorylated tyrosine in the C-terminus (Tyr527) c-Abl achieves its autoinhibition by binding of the myristoyl group in a hydrophobic pocket inside the kinase domain In the inactive conformation of c-Src and c-Abl, the SH2 and SH3 domains pack against the kinase domain The activation of c-Src and c-Abl involves three steps: unlatching, unclamping, switching Imatinib (STI-571) can effectively inhibit c-Abl, as the kinase domain exhibits a unique conformation of the DFG-activation loop... next topic---glycoproteins Medizinische Fakultät 25. November 2024 31 Glycosylation is a highly regulated process that attaches several of ninebase monosaccharides to proteins and lipids forming diverse biopolymers involvingenzymes such as glycosyltransferase andglycosidase.  Glycosyltransferases: synthesise glycan chains  Glycosidases: hydrolyse specific glycan linkages and break down glycans or glycoproteins Glycan can be attached in different ways to protein. The most common are:  N-glycans – linked to a N-atom of the amide group in the Asn of Asn-X-Ser/Thr where X ≠ proline  O-glycans –linked to an O-atom of the hydroxyl group in the subset of Ser and Thr  Glycosaminoglycans(GAGs)/Proteoglycans – also linked to Ser and Thr residues, but are linear and highly sulphated Glycosylation isregulatedonmultiplelevelsinvolvingposttranscriptionalandposttranslationalmechanisms: G lycosyltransferaseandglycosidaseexpression(RNAexpression),accessibilitytosubstratesandenzymatica ctivity Glycans have multiple different functions:  cell adhesion, self-/non-self-recognition, molecular trafficking and clearance ofprotein,receptoractivationandendocytosis,structuralsupportandproteinfolding Take Home Messages Medizinische Fakultät 25. November 2024 32 Take Home Messages Glycans provide structural support by N-linkedglycosylation.Thisincludes: A ssistinginproteinfolding,stabilisingmatureproteins,aidingintheassemblyofoligomericcomplexesanden ablingcellsurfaceglycoproteinstoorientthemselvesonthecellsurface Understanding the structureand targetsof carbohydrate-recognising proteins (e.g. lectins or adhesins) in pathogens and physiological processes offers an approach for the development of new inhibitors and drugs. Th is includesfollowing structures and their corresponding glycan targets :  UDP-galactopyranose mutase : D-galactofuranose(Galf)  Haemagglutinin-neuraminidase (HN) : terminal sialic acid residues  Staphylococcal superantigen-like proteins SSL : sialylatedglycoproteins on cell surfaces (SSL11–SLeXcomplex) S iglecs/Galectinsin human cell-cell interactions: Sialic acids (CD22) and β-galactoside(galectin3) The human A, B, and H antigens determine the histo-blood groups A,B,O. They are complex fucosylatedoligosaccharides with differences in side-chains thatare selectively bound by pathogens. This can be demonstrated by two noroviruses:  Norwalk virus binds only A antigen(GalNAc)  VA387 bind swith A antigen (GalNAc)andBantigen (galactose)...next topic....protein dynamics Take-home messages I MD simulations allow to study biological properties that can hardly be deduced from a static picture alone, e.g.  Protein folding and stability  Affinity of substrate and ligand binding  Conformational changes relevant for molecular recognition processes  Ion transport through ion channels MD simulations are computationally expensive, because  fast vibrational motions require small time steps  long-ranging electrostatic interactions need to be considered  long simulation times required to cover relevant biological motions  explicit treatment of the solvent (> 100.000 atoms) required  multiple simulations required to enhance statistical significance  multiple simulations required to investigate different mutations and ligands MD simulations...  are based on Newton‘s equation of motion  use a potential function to describe covalent and noncovalent interactions  describe covalent interactions by harmonic potentials  generate a trajectory by integrating Newton’s equation of motion For different types of biological motions the minimal simulation time required is  Bond vibrations > 10-15 s (1 fs)  Collective vibrations, side chain motions > 10-12 s (1 ps)  Helix/domain/subunit motions > 10-9 s (1 ns)  Helix/coil transition > 10-7 s (100 ns)  Dissociation/association, ligand binding > 10-7 s (100 ns)  Protein (un)folding > 10-6 s (1 μs) Take-home messages II...next topic...potasium ionchannels..Potassium channels are critical for maintaining the membrane potential and ionic balance in excitable (e.g. neurons, muscles) & non-excitable cells Selectivity filter: 10 000-fold preference for K⁺ over Na⁺ Precise binding site geometry & energetic balance during ion dehydration and rehydration Firm packing of side chains → carbonyl oxygens can`t get close enough to compensate for cost of hydration of smaller Na+ ions (energetically unfavorable) Potassium channels achieve near diffusion limit rates of ion conduction Two K+ ions at each end of the selectivity filter → Movement of K+ ions by mutual repulsion & membrane potential Arrangement of oxygens mimics water oxygens surrounding a K+ ion in solution→ low transfer energy Gating is controlled through intracellular and extracellular mechanisms  Extracellular inactivation: N-type inactivation (fast, autoinhibitory peptide occlusion) C-type inactivation (slower, conformational changes in the selectivity filter) Different classes of potassium channels (voltage-gated, inward rectifiers, tandem pore domain, ligand-gated) exhibit structural variations to adapt to unique physiological roles Kv channels important in neuronal excitability → restore membrane potential after AP Voltage-sensing domain (S1–S4 helices) triggers conformational changes, gating the pore-forming domain (S5–S6 helices) Gating of Kv channels is both lipid- and voltage-dependent...next topic...gpcr rhodopsin....1. Light triggers the isomerization of the retinal C11=C12 double bond from cis to trans. 2. Retinal isomerization leads to steric strain within the retinal binding site and internal proton transfer from the retinal protonated Schiff base to a glutamate. 3. Sequence motifs that are important for the activation of rhodopsin are the (E/D)RY motif and the NPxxY motif. 4. The metarhodopsin II state binds and activates transducin. 5. Three levels of conservation can be found within class A of GPCRs: signature amino acids, group-conserved residues and subfamily-specific residues. 6. Mutations of amino acids can lead to constitutive activity of rhodopsin. Certain mutations cause congenital night blindness and autosomal dominant retinitis pigmentosa. Short linear motifs (SLiMs) in protein-protein interactions....Take-Home-Messages I Protein interaction domains (PIDs)... - are relatively small (~ 100 amino acids) - are of modular nature, with amino- and carboxy-termini in close spatial proximity. - are present in many different kinds of proteins - mediate interactions with other proteins by recognizing short sequence motifs - frequently recognize post-translational modifications Short linear sequence motifs (SLiMs)... - are disordered/flexible prior to binding - are ~ 3-10 amino acids long - are frequently regulated by posttranslational modifications (PTMs) - mediate protein binding, targeting, cleavage, PTMs - frequently exhibit low binding affinity and specificity - are frequently observed in signaling networks - are described as sequence patterns (‘regular expressions’) Take-Home-Messages II Typical pairs of PIDs and SLiMs are: - SH3 domains bind proline-rich peptides - PDZ domains bind the C-terminus of peptides - SH2 domains bind phosphotyrosine-containing peptides - 14-3-3 domains bind phosphoserine-containing peptides - Chromo domains bind methyllysine-containing peptides - Bromo domains bind acetyllysine-containing peptides Physiological importance of PID-SLiM interactions: - PID-SLiM interactions allow for complex signaling cascades (including inducibility, cooperativity, sequential or antagonistic regulation) - Some cellular processes like vesicle trafficking are almost exclusively guided by SLiMs - Mutations in SLiMs frequently cause diseases - Viral proteins frequently mimic SLiMs to interfere with the cellular machinery (e.g. signaling, targeting, transcription,...).....next topic Protein modules mediating RNA-Recognition 3. Januar 2025 18 Take home messages (I) Structural properties of ssRNA binding domains Principles of ssRNA recognition proteins 2typesofrecognitionpossible:ConservedRNAbindingdomains(RRM,KHandOB- fold)andmodularRNAbindingrepeatsoroligomers(OB-fold,TRAP,SmproteinsandPUM-HD). 2-10ntsperdomainormonomer.Usuallyin5’/3’toC-/N-terminaldirection. Hugeversatility:greatvarietyofproteinfolds(β-sheetsurfaces,α- helicalfoldsorloops),posibleinteractions(hydrophobic/stackinginteractionsandhydrogenbonds)andsel ectivityforRNAvsDNA Conformationalflexibility:eitherthessRNAortheproteincanchangeshapeinordertofittoeachother. RRMdomains:  4strandedantiparallelβ-sheetpackedagainsttwoα-helices. StackinginteractionsofaromaticsidechainswithRNAbases Alltheelementsinthestructureinteract,buttheβ-sheetistheprimaryRNAbindingsurface KHdomains: Three-strandedβ-sheetpackedagainstthreeα-helicesTwofoldvariantsaroundacommonβααβcore Hydrophobicinteractions Medizinische Fakultät 3. Januar 2025 19 Take home messages (II) Structural properties of ssRNA binding domains Medizinische Fakultät im Medizincampus Oberfranken OB-fold: Five-strandedβ-barrelarrangedinaGreekkeymotif Hydrophobic/aromaticinteractionsandhydrogenbonds. TRAP: Antiparallelβ-sandwichfoldwiththetryptophaninsertedbetweenthetwoβ- platforms11subunitsassembleinasymmetricring,inwhichthessRNAbindontheoutersurface Hydrogenbonds Smfold: α-helixfollowedbyafive-strandedantiparallelβ- sheetstronglybent7SMproteinsassembleinanoligomericring- likestructure,inwhichssRNAbindintheinnersurface Stackinginteractionsandhydrogenbonding. PUMhomologydomain Pufrepeats:Three- helicalbundlewithoneshorthelix(α1)andtwolonghelixes(α2andα3)curvedstructureof8consecutivePuf repeats stackinginteractionsandhydrogenbonds. 3. Januar 2025 20 Take home messages (III) RRM versatility HighdiversityofRRM–ligandrecognition. RRMscaninteractwithnucleicacid,withthemselvesandwithotherproteins.  β-sheetbindsRNAnotalwayssequence-specifically. Notonlytheβ-sheetsurfacebutalsotheloopsconnectingβ-strandsandα- helicescanbecrucialfornucleicacidrecognitionRRMsinteractusingalltheelementscomposingtheirstruc ture. Conservedaromaticresiduesareimportantfordifferentinteractions. RRM-RRMinteractionscanincreasetheRNAaffinity,createloopsintheRNAorpreventtheRNAbinding- RRM-proteininteractionscanbothallowornotthesimultaneousinteractionwithRNA.....next topic...antibody antigen recognition...Medizinische Fakultät 2. Dezember 2024 15 Antibody diversity is achieved via somatic recombination of AB encoding genes (IGHV, IGHD, IGHJ, IGLV, IGLJ ,IGKV, IGKJ) and resulting combinatorial diversity; diversity further enhanced by junctional diversity and somatic hypermutation Antigen specificity is mainly determined by 6 hypervariable regions (CDR) within variable domains (3 in HC, 3 in LC); HC CDR3 most diverse, in most cases largest contributor to ABS Hypervariable regions form surface of antigen binding site (paratope) that is extremely specific to antigen (epitope) Antibody contact residues on ABS interact with antigen contact residues via chemical attraction forces (VdW, ionic bonds, hydrogen bonds) non covalently Small molecules called haptens bind in crevices of the antibody surface; haptens alone have no immunogenicity; antibodies can have catalytic activitywith haptens as substrates MHC I located on all cell types, responsible for recognition of self and of intracellular pathogens toCD8 T-cells MHC II located on B-cells and macrophages, responsible for presenting pathogen derived peptidestoCD4 T cells MHC molecules’ peptide binding site is lined by two 𝝰𝝰-helices and binding specificity is determined by amino acid sequence MHC molecules are extremely variable in humans and specifically bind short peptide antigens (~7- 14 residues in MHC I, ~9-30 in MHC II) in binding crevice  T cell receptor sonly recognizes antigens when attached to MHC molecule Take home message next topic Recognition of HIV-1 gp120 by antibodies Medizinische Fakultät 15 HIV-1 has challenging properties, which prevent creation of AIDS-vaccine −diversity of HIV-strains −evasion of immunsystem −Immunosuppression −incorporate viral genom in host DNA HIV-1 antibodies need to fulfill specific criteria −broadly neutralizing Ab (bnAb) −specificity (no auto reactivity) −high enough titer in human promising antibodies target CD4-binding-site (CD4bs) of gp120, e.g. VRC01 VRC01 binding to gp120 −main interaction site: CDR H2 of heavy chain −binding site: Arg71 as dominant residue and three hydrogen bonds Take-home message 1 02. Dezember 2024 Medizinische Fakultät 16 VRC01 mimics CD4 binding, but has considerable differences −similar binding to gp120 VRC01 mimics one of two important residues of CD4 (Arg71) three hydrogen bonds −different orientation: VRC01 rotates 43°relative to the CD4-defined orientation and translates 6 Å away from the bridging sheet clash-free orientation VRC01 has a high recognition diversity because it avoids conformational masking and glycan shielding natural resistance exists due to variations at the tip of the V5 loop of gp120 but observedresistance frequency is smaller than expected −V5 loop fits into gap between heavy and light chains −VRC01 contacts only the more conserved residues at the loop base −VRC01 tolerates variations in the tip of the V5 loop Take-home message 2 next topic--viral fusion proteins Fusion proteins: expressed on viriom surface, facilitate membrane fusion during invasion Priming: irreversible change as preparation for mediating membrane fusion Triggering: initiation of conformational transition for membrane fusion Membrane fusion steps: (Receptor binding)  Connection of membrane through viral membrane protein  Approximation of membranes  Hemifusion  Fusion pore  Fused membranes HisCat-Interactions: Interaction between Histidine and nearby cationic residue (Lys, Arg, His)  Decrease in pH leads to electrostatic repulsion AniAni-Interactions: Interaction between two anionic residues (Asp or Glu) in close proximity  Neutral pH: electrostatic repulsion (aleviated on pH decrease) Three classes of viral membrane fusion proteins: Class I: Priming by cleavage of trimeric protein Examples: Influenza virus, HIV Class II: Priming by cleavage of chaperone Examples: Flavivirus (Dengue virus), Alphaviruses Class III: No priming, reversible triggering Examples: VSV The glycoprotein G of VSV transitions reversibly between pre- and post-fusion states, allowing it to navigate acidic Golgi compartments without functional loss. This unique capability distinguishes it from other viral fusion proteins. G operates in a pH-dependent dynamic equilibrium among prefusion, activated, and post-fusion states. The prefusion state stabilizes the viral surface, while the post-fusion state facilitates membrane integration, showcasing a distinct fusion mechanism. With characteristics of both Class I and Class II fusion proteins, G may define a new class. Its exposed fusion loops and lack of proteolytic activation challenge traditional categorizations and expand insights into viral entry. Conserved residues in G act as pH-sensitive switches, where histidine protonation triggers fusion activation at low pH, and deprotonation restores the prefusion state. This ensures adaptability and precise regulation. Fusion intermediates (VSV) rely on cooperative action by ~15 G trimers to overcome the significant energy barrier of pore formation, emphasizing the complexity of its fusion mechanism. next topic--The SARS CoV 2 spike protein 22 Take home messages 1. Homotrimeric spike protein drives SARS CoV 2 infection by binding to ACE2 receptor on a host cell and mediates membrane fusion, making it a prime target for vaccines and therapies 2. The Spike protein resembles type 1 viral fusion proteins and requires activation by host enzymes to mediate infection. 3. S protein has two subunits: receptor binding fragment S1 (consists of NTD, CTD1&2 and RBD) and fusion fragment S2 (consists of FP, HR1&2 and others). 4. RBDs adopt two conformations: “up”(accessible state) and “down” (receptor inaccesible state). 23 Take home messages 1. Class 1 antibodies have a short CDRH3 loop that allows them to bind only to the “up” conformation of RBDs. 2. Class 2 antibodies can bind to both “up” and “down”. They have a longer CDRH3 loop. They can cross link between different RBDs based on their conformation. 3. Class 3 antibodies bind the RBDs in a site that does not overlap with ACE2 binding site and recognise both “up” and “down” RBDs. 4. Class 4 antibodies don’t block ACE2 and only bind to “up” RBDs 5. Combining different classes of neutralzing antibodies is a promisingtherapeutic strategy to achieve broader protection against variants. 6. Mutations affecting one class of NAbs do not necessarily impact other classes...next topic...Protein folding and flexibility Medizinische Fakultät 13. Januar 2025 27 The thermodynamic stability of a protein in its native state is low and depends on the differences in entropy and enthalpy between the native state and the unfolded state. It is important that this free energy difference is small (5 15 kcal/mol) because cells must be able to degrade and synthesize proteins quickly. Upon folding a polypeptide chain, hydrophobic residues tend to be buried in the interior, greatly restricting the number of possible conformations the chain can assume, and therefore allowing proteins to fold in seconds rather than years. Within milliseconds the polypeptide chain achieves the molten globule state, a set of structures that have in common a loosely packed hydrophobic core and some secondary structure. Some proteins have one preferred folding pathway, while others seem to have multiple parallel pathways to the native state. Cells contain enzymes such as cis trans proline isomerases/PDI that catalyze otherwise long lasting processes. Unfolded proteins with exposed hydrophobic patches aggregate easily by non specific hydrophobic interactions. To circumvent this problem a class of proteins called chaperones have evolved to sequester unfolded polypeptides. Take home messages I Medizinische Fakultät 13. Januar 2025 28 The chaperonins GroEL (two heptameric rings, 547 aa per subunit) and GroES (one heptameric ring) form short cylinders which, because they have hydrophobic residues in the interior, can bind to any unfolded polypeptide with exposed hydrophobic patches, regardless of its amino acid sequence. Once the polypeptide chain is in the chaperonins it is protected from aggregation with other proteins. After iterative rounds of folding and releasing (under ATP hydrolysis), the native state can be reached. Even inside crystals all atoms in protein molecules undergo small oscillations (breathing). In addition to breathing, some proteins undergo large conformational changes in response to ligand binding or to changes in their environment, and these conformational changes are essential for function. CDKs are crucial for the control of the cell cycle. CDK2 undergoes changes in the conformation of the PSTAIRE helix and the flexible T loop region (upon interaction with the cyclin box of Cyclin A), while the complex partner Cyclin A stays virtuall y the same. The serpins, serine proteinase inhibitors, undergo drastic conformational change: activation of the inactive form involves conversion of a β strand in the middle of a β sheet into a flexible loop and the converse occurs when the active form changes into the latent form. PFK is subject to allosteric control and exists in two states, the R (relaxed) and the T (tense) state. Effector molecules ha ve a high affinity for only one of these states. Therefore, when an effector molecule is present, it shifts the equilibrium to fav or the high affinity state (F6P and ADP shift to the active R state, PEP shifts to the inactive T state). The active site in the R stat e has a thousandfold higher affinity for the substrate F6P than does the active site of the T state, due to structural differen ces between the active sites in these two states. Take home messages II next topic...protein degradation.. Medizinische Fakultät January 10, 2025 34 The 20S proteasome is a core component of the proteasome complex, which is crucial for protein degradation. It’s composed of 4 stacked rings, each consisting of 7 subunits. The two outer rings are made of α subunits while the two inner rings are made of β subunits. α1-7, β1-7, β1-7, α1-7 stoichiometry in eukaryotic cells. The α and β subunits are different in prokaryotes and eukaryotes. The 20S proteasome has a molecular dimension of 160 Å in length and 120 Å in diameter. The α rings form a gate-like structure and control the access to the proteolytic chamber with the N-terminal regions. The YDR motif is key for the regulatory gating process. They are present in all organisms but with different tertiary structures and functions between eukaryotes and prokaryotes. The β rings contain the catalytic sites. The eukaryotic only have 3 proteolytically active subunits (β1, β2 and β5), and they perform different types of proteolytic cleavage. The catalytic sites are characterized by the presence of a catalytic triad formed with histidine, serine and asparagine residues. The proteolytic active centers are formed by the N-terminal threonine of each β subunit. The 26S proteasome is formed by one 20S proteasome and two 19S regulatory cap subunits. The 19S confer ATP- and ubiquitin-dependence on proteolysis. Take home messages part I Medizinische Fakultät January 10, 2025 35 Take home messages part II HslVU: Composed of HslV (hexameric dimer) and HslU (hexamer), functioning as an ATP-dependent protease.  ATP binding to HslU induces conformational changes that activate HslV to degrade substrates. Maintains bacterial proteostasis by targeting misfolded proteins for degradation and is found in both bacteria and primordial eukaryotes. Tricorn Protease: A homohexameric protease (part of a bigger structure) with a 14.6 MDa capsid, featuring β6 and β7 propeller domains. Processes substrates through the β7 propeller (entry) and releases products via the β6 propeller (exit). Functions downstream of the proteasome, cleaving peptides into amino acids, often with the help of accessory proteins like F1, F2, and F3. DegP: Forms hexameric or dodecameric cage-like complexes with temperature-sensitive flexibility. Exhibits dual roles: Low temperature: Acts as a chaperone to stabilize misfolded proteins. High temperature: Switches to a protease to degrade misfolded proteins using its catalytic triad (serine-histidine-aspartate). A vital component of bacterial stress response, making it a potential antibiotic target...next topic..fibrous proteins...Take home messages Medizinische Fakultät 20. Januar 2025 16 1. Fibrous proteins are molecules with long polypeptide chains that can be separated into three assembled groups: coiled coil α hel ices, triple helix, β sheets 2. Collagen assembles a right handed superhelix containing repeat sequences (often Gly Pro Hyp ). Important for the stability are hydrogen bonds of proline C=O and glycine NH of two different chains and water mediated hydrogen bonds between OH group of Hyp and peptide groups. 3. F actin is a right handed helical polymer of G actin monomers. The assembling is ATP mediated 4. Myosin consists of two heavy chains , which form the coiled coil tail and the globular heads , and of four light chains bound to the globular heads 5. The swinging cross bridge model explains the muscle contraction without shortening of the filaments. Actin and myosin glide over each other. 6. ATP and actin binding at the cross bridge of myosin is strong negative coupled a) Rigor state : absence of nucleotide and strong binding to actin b) Post rigor state : binding ATP and releasing actin c) Return stroke : ATP hydrolysis and conformational change of the lever arm to up position d) Pre power stroke state : weak binding to actin and release of the γ phosphate e) Power stroke : Release of ATP, strong binding to actin and conformational change of the lever arm to down position  movement of 80 100 Å (per cycle)...last topic....protein aggregation....Medizinische Fakultät 20. Januar 2025 47  Amyloid Definition and Structure Amyloids are elongated unbranched fibers with cross β structures formed through backbone hydrogen bonding in proteins Their stability arises from structures like steric zippers where β sheets interlock tightly  Formation and Mechanisms Amyloidogenesis involves nucleation elongation and fragmentation often influenced by protein concentration mutations and cellular conditions Cross seeding and cross inhibition mechanisms highlight interactions between different amyloid types  Pathological and Functional Roles Pathological amyloids such as A β in Alzheimer’s and tau in Parkinson’s contribute to cellular toxicity via oligomers and fibrils Functional amyloids exist in controlled biological roles e g hormone storage and immune responses  Strain Diversity and Disease Amyloid strains arise from structural polymorphisms with distinct morphotypes influencing disease manifestation and progression Environmental and genetic factors contribute to strain variability  Transmissibility Amyloid diseases like prion disorders exhibit transmissibility through seeding though human to human transmission remains rare Take Home Messages I Medizinische Fakultät 20. Januar 2025 48 Amyloid β Aggregation  Amyloid β is a peptide of 39 42 amino acids in the brain with Aβ 1 40 and Aβ 1 42 as the most abundant isoforms Aβ aggregates, especially small oligomers are neurotoxic and therefore can cause the neurodegenerative Alzheimer Disease („amyloid hypothesis  Aβ monomers exist in an equilibrium between an α helical and an β sheet conformation Aβ protofibrils consist of a repetitiv structure of these β sheet rich monomers (cf Take Home Figure)  The Aβ aggregates Protofibrils Fibrils Plaques) assemble by seeded growth The rate limiting step for that is the build up of oligomers that function as seeding nuclei Prion Diseases  Prion diseases are transmissible neurogenerative disorders that are caused by missfolded aggregated Prion Proteins PrP  By Seeded Polymerization newly bound PrP sen (native conformation transforms into β sheet rich insoluble PrP res pathological conformation  Examples for Prion diseases are the Creutzfeldt Jakob disease ( in humans the bovine spongiform encephalopathy ( in cattle the chronic wasting disease ( in deers elk and scrapie in sheep Take Home Messages II