Protein Structure 2 Protein Modification BL4010 10.03.06

Nomenclature Domain - a particular region within a polypeptide chain (ATP binding domain) Motif - a protein structural element (may appear more than once in a single protein or in many different types of proteins (e.g. greek key) Subunit - a single polypeptide unit within a larger multipeptide protein Complex - group of proteins with long-term or transient physical association

Multidomain proteins

Multisubunit proteins

Quaternary (4°) structure What are the forces driving quaternary association? Typical Kd for two subunits: 10-8 to 10-16M! These values correspond to energies of 50-100 kJ/mol at 37 C Entropy loss due to association - unfavorable Entropy gain due to burying of hydrophobic groups - very favorable!

Alcohol dehydrogenase dimer

Prealbumin dimer

Tyrosine kinases

Packing symmetry

Multiple subunits vs. multiple domains Structural factors Stability: reduction of surface to volume ratio Bringing catalytic sites together (efficiency) Flexibility in shared binding sites Increased complexity Genetic factors Gene duplication = genetic freedom/economy Multiple interactions = increased impact of mutation Regulation Cooperativity Must be co-expressed

Tubulin as an example Essential component of the cytoskeleton Dimers polymerize Multiple sites of interaction Dynamic Mutations have severe effects

Protein Modification

Post-translational Modifications Cleavage of signal peptides Phosphorylation Amidation Glycosylation Hydroxylation Ubiquitination Addition of prosthetic groups Iodination Adenylation Sulfonation Prenylation Myristoylation Acylation Acetylation Methylation Oxidative crosslinking N-Glutamyl cyclization Carboxylation Table 4.1

Are disulfide bonds post-translational modification?

N-terminal modifications N-formyl Met (by product of translation process and incomplete cleavage by deformylase) Aminopeptidase cleavage of Met N-terminal acetylation N-terminal myristoylation N-terminal glutamyl cyclization

C-terminal modification C-terminal prenylation farnesylation geranylation/geranyl-geranylation C-terminal amidation

Many (but not all) post-translational modifications involve parts of the secretion apparatus

Phosphorylation Hydroxyl groups - Ser, Thr, Tyr Amide groups of His, Lys & Carboxy group Asp Often causes changes in protein conformation and/or protein-protein interactions

C-terminal Amidation Neuropeptides and hormones C-terminal glycine is hydroxylated alpha-hydroxy-glycine. Glyoxylate. C-terminal amidation is essential to the biological activity of many neuropeptides and hormones.

Hydroxylation and Carboxylation Hydroxylation (previous lecture) Lys and Pro (e.g. collagen) Requires vitamin C Carboxylation Requires viatamin K

Iodination Special case: Tyr  thyroxin

Protein Acylation Methylation and acetylation S-acylation (Cys) acetylation of histone Lys S-acylation (Cys) Prenylation (C-terminus) Myristoylation (N-terminus)

Acetylation

N-terminal acetylation No simple sequence consensus Usually accompanied by cleavage of the N-terminal Met

Mass spectral analysis

Methylation Can occur at carboxyls, amides, sulfhydryl, etc. SAM is the activated carbon source

Multiple methylation

Histones DNA binding proteins Chromatin structure Affect gene expression

Sulfonation O-sulfonation of Ser/Thr discovered in 2004 by MS analysis (Mol Cell Proteomics. 3(5):429-40)

N-terminal myristoylation Consensus Gly-X-X-X-Ser/Thr myristoyl Coenzyme A

C-terminal Prenylation methylated c-terminus C-terminal Prenylation Prenylation CAAX motif AAX cleaved and Cys methylated then prenyl group added by thioether linkage (C-S-C). n-terminus

S-acylation

Adenylation Toxins frequently adenylate host target proteins to inactivate them Glutamine synthetase is inactivated by adenylylation and activated by deadenylylation

Adenylation Adenylation of a DNA ligase Lys residue is a key step in DNA replication and repair

All non-cytoplasmic proteins must be translocated Proteolytic cleavage All non-cytoplasmic proteins must be translocated The leader peptide retards the folding of the protein so that molecular chaperone proteins can interact with it and direct its folding The leader peptide also provides recognition signals for the translocation machinery A leader peptidase removes the leader sequence when folding and targeting are assured

Proteolytic processing Proteolytic cleavage of the hydrophobic N-terminal signal peptide sequence Proteolytic cleavage at a site defined by pairs of basic amino acid residues Proteolytic cleavage at sites designated by single Arg residues Signal prediction algorithms (e.g. SignalP) caution: may predict TMDs as signalPs

Hormones All secreted polypeptide hormones are synthesized with a signal sequence (which directs them to secretory granules, then out) Usually synthesized as inactive preprohormones ("pre-pro" implies at least two processing steps) Proteolytic processing produces the prohormone and the hormone

GASTRIN Product of preprogastrin Signal peptide cleavage 101-104 residues Signal peptide cleavage prepropeptide  propeptide 80-83 residues Cleavage at Lys and Arg residues and C-terminal amidation leaves gastrin propeptide  peptide N-terminal residue of gastrin is pyroglutamate C-terminal amidation involves destruction of Gly

GASTRIN

GASTRIN Heptadecapeptide secreted by the antral mucosa of stomach stimulates acid secretion in stomach

Glycosylation: 2 flavors O-linked GalNac (Ser, Thr, Hyp) N-linked GlcNAc (Asn)

Glycosylation

Protein glycosylation Usually extracellular or at cell surface High structural information content molecular recognition Occurs along the secretory pathway Often stabilizes structure Difficult to get crystal structure for more than one or two carbohydrate residues

Protein glycosylation

O-linked glycosylation No consensus sequence for Ser and Thr Consensus for Hyp Gly – X – Hyl – Y – Arg Begins with GalNac transferase (N-acetylgalactosamine) Mannose common addition to core

The ABO(H) blood group determinant is an O-linked polysaccharide In 1901, Karl Landsteiner discovered blood group antigens. Since that time nearly 200 antigens have been identified. Carbohydrate-dependant blood group antigens are carried by both glycoproteins and glycolipids

N-linked glycosylation Most proteins contain several potential sites but usually only a few (in any) are actually glycosylated Many different patterns Consensus (-N-X-T/S-) Copyright 2000-2002 IonSource.Com

N-linked glycosylation begins in the cytoplasm glycosyl-dolichol-phosphate (lipid) core is transferred to protein

Further processing and elaboration occurs in the ER and Golgi

Carbohydrates are flexible Variety of conformations difficult to get good x-ray data Structural information H-bonding hydrophobic patches wheat germ agglutinin

GPI anchors (glycosylphosphatidyl inositol)

Carbohydrates as cross-linkers Galectin-1 dimer

Ubiquitination DEGRADATION

Ubiquitination Ubiquitinating enzymes E1,E2, E3 - thiol ester bond Final target - isopeptide bond between a lysine residue of the substrate (or the N terminus of the substrate) and ubiquitin Ubiquitin first activated by adenylation Science 2005 309:127-130 target protein ubiquitination on Cys!

More about proteoglycans and ubiquitination later