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Protein Folding, Binding and Evolution: PDZ domains and paralemmins as model systems
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. (Per Jemth)
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins present at the synapse need to be multitasking in order to perform all vital functions in this limited space. In this thesis I have analyzed the function and evolution of such proteins, focusing on the PDZ domain and the paralemmin family. The PDZ domains bind to a wide variety of interaction partners. The affinity for each partner is regulated by residues at the binding site, but also through intradomain allostery. How this intradomain allostery is transferred to the binding site is not established. I here show that side chain interactions can explain all transfer of intradomain allostery in three analyzed PDZ domains. A circularly permuted PDZ domain has an identical set of amino acids as the original protein and a very similar structure with only a few perturbed side chains. By using the circular permutant I show that a slight alteration in the position of a side chain leads to a corresponding change in allosteric signal. I further study the folding of several PDZ domains and show that they all fold via a conserved folding mechanism, supporting the notion that the final structure has a part in deciding folding mechanism. The folding mechanism of the circularly permuted PDZ domain is conserved compared to the original protein illustrating how circular permutations can be tolerated through evolution. The multifunctionality of paralemmins probably lies in their highly flexible structures. I have studied the evolution of the paralemmins and found that the four mammalian paralemmins arose in the two whole-genome duplications that occurred early in the vertebrate evolution. The fact that all four paralemmins have survived evolution since the gene duplications suggests that they have important functions, possibly in the development of the nervous system. Synaptic proteins are crucial for many biological processes, and their misfolding implicated in many diseases. The results presented here shed light on the mechanisms of action of the synaptic proteins and will help us to understand how they generate disease.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. , 47 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1006
Keyword [en]
Protein folding, evolution, binding, allostery, Paralemmin, PDZ domain
National Category
Biochemistry and Molecular Biology Structural Biology Genetics
Identifiers
URN: urn:nbn:se:uu:diva-185573ISBN: 978-91-554-8563-4 (print)OAI: oai:DiVA.org:uu-185573DiVA: diva2:575642
Public defence
2013-02-01, B42, BMC, Husargatan 3, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2013-01-11 Created: 2012-11-26 Last updated: 2013-02-11Bibliographically approved
List of papers
1. An expanded view of the protein folding landscape of PDZ domains
Open this publication in new window or tab >>An expanded view of the protein folding landscape of PDZ domains
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2012 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 421, no 3, 550-553 p.Article in journal (Refereed) Published
Abstract [en]

Most protein domains fold in an apparently co-operative and two-state manner with only the native and denatured states significantly populated at any experimental condition. However, the protein folding energy landscape is often rugged and different transition states may be rate limiting for the folding reaction under different conditions, as seen for the PDZ protein domain family. We have here analyzed the folding kinetics of two PDZ domains and found that a previously undetected third transition state is rate limiting under conditions that stabilize the native state relative to the denatured state. In light of these results, we have re-analyzed previous folding data on PDZ domains and present a unified folding mechanism with three distinct transition states separated by two high-energy intermediates. Our data show that sequence composition tunes the relative stabilities of folding transition states within the PDZ family, while the overall mechanism is determined by topology. This model captures the kinetic folding mechanism of all PDZ domains studied to date.

Keyword
PDZ folding transition state
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-185575 (URN)10.1016/j.bbrc.2012.04.042 (DOI)000306152000025 ()22521641 (PubMedID)
Available from: 2012-11-26 Created: 2012-11-26 Last updated: 2017-10-16Bibliographically approved
2. Tolerance of Protein Folding to a Circular Permutation in a PDZ Domain
Open this publication in new window or tab >>Tolerance of Protein Folding to a Circular Permutation in a PDZ Domain
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 11, e50055- p.Article in journal (Refereed) Published
Abstract [en]

Circular permutation is a common molecular mechanism for evolution of proteins. However, such re-arrangement of secondary structure connectivity may interfere with the folding mechanism causing accumulation of folding intermediates, which in turn can lead to misfolding. We solved the crystal structure and investigated the folding pathway of a circularly permuted variant of a PDZ domain, SAP97 PDZ2. Our data illustrate how well circular permutation may work as a mechanism for molecular evolution. The circular permutant retains the overall structure and function of the native protein domain. Further, unlike most examples in the literature, this circular permutant displays a folding mechanism that is virtually identical to that of the wild type. This observation contrasts with previous data on the circularly permuted PDZ2 domain from PTP-BL, for which the folding pathway was remarkably affected by the same mutation in sequence connectivity. The different effects of this circular permutation in two homologous proteins show the strong influence of sequence as compared to topology. Circular permutation, when peripheral to the major folding nucleus, may have little effect on folding pathways and could explain why, despite the dramatic change in primary structure, it is frequently tolerated by different protein folds.

Keyword
Circular permutant, protein folding, PDZ domain
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-185577 (URN)10.1371/journal.pone.0050055 (DOI)000311821000173 ()
Available from: 2012-11-26 Created: 2012-11-26 Last updated: 2017-12-07Bibliographically approved
3. Energetic pathway sampling in a protein interaction domain
Open this publication in new window or tab >>Energetic pathway sampling in a protein interaction domain
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2013 (English)In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 21, 1193-1202 p.Article in journal (Other academic) Published
Abstract [en]

The affinity and specificity of protein-ligand interactions are influenced by energeticcrosstalk within the protein domain. However, the molecular details of such intradomain allostery are still unclear. Here, we have experimentally detected and computationally predicted interactionpathways in the postsynaptic density 95/discs large/zonula occludens 1 (PDZ)-peptide ligand model system using wild-type and circularly permuted PDZ proteins. The circular permutant introduced small perturbations in the tertiary structure and a concomitant rewiring of allosteric pathways, allowing us to describe how subtle changes may reshape energetic signaling. The results were analyzed in the context of other members of the PDZ family, which were found to contain distinct interaction pathways for different peptide ligands. The data reveal a fascinating scenario whereby several energetic pathways are sampled within one single domain and distinct pathways are activated by specific protein ligands. 

Keyword
intradomain allostery, PDZ domain, protein binding, circular permutant
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-185579 (URN)10.1016/j.str.2013.05.010 (DOI)000321681600016 ()
Available from: 2012-12-10 Created: 2012-11-26 Last updated: 2017-10-16Bibliographically approved
4. Evolution of the Vertebrate Paralemmin Gene Family: Ancient Origin of Gene Duplicates Suggests Distinct Functions
Open this publication in new window or tab >>Evolution of the Vertebrate Paralemmin Gene Family: Ancient Origin of Gene Duplicates Suggests Distinct Functions
2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 7, e41850- p.Article in journal (Refereed) Published
Abstract [en]

Paralemmin-1 is a protein implicated in plasma membrane dynamics, the development of filopodia, neurites and dendritic spines, as well as the invasiveness and metastatic potential of cancer cells. However, little is known about its mode of action, or about the biological functions of the other paralemmin isoforms: paralemmin-2, paralemmin-3 and palmdelphin. We describe here evolutionary analyses of the paralemmin gene family in a broad range of vertebrate species. Our results suggest that the four paralemmin isoform genes (PALM1, PALM2, PALM3 and PALMD) arose by quadruplication of an ancestral gene in the two early vertebrate genome duplications. Paralemmin-1 and palmdelphin were further duplicated in the teleost fish specific genome duplication. We identified a unique sequence motif common to all paralemmins, consisting of 11 highly conserved residues of which four are invariant. A single full-length paralemmin homolog with this motif was identified in the genome of the sea lamprey Petromyzon marinus and an isolated putative paralemmin motif could be detected in the genome of the lancelet Branchiostoma floridae. This allows us to conclude that the paralemmin gene family arose early and has been maintained throughout vertebrate evolution, suggesting functional diversification and specific biological roles of the paralemmin isoforms. The paralemmin genes have also maintained specific features of gene organisation and sequence. This includes the occurrence of closely linked downstream genes, initially identified as a readthrough fusion protein with mammalian paralemmin-2 (Palm2-AKAP2). We have found evidence for such an arrangement for paralemmin-1 and -2 in several vertebrate genomes, as well as for palmdelphin and paralemmin-3 in teleost fish genomes, and suggest the name paralemmin downstream genes (PDG) for this new gene family. Thus, our findings point to ancient roles for paralemmins and distinct biological functions of the gene duplicates.

Keyword
genome duplication, molecular characterization, chromosome duplications, membrane dynamics, amphioxus genome, teleost fishes, protein family, insights, reveals, identification
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-184483 (URN)10.1371/journal.pone.0041850 (DOI)000306806600134 ()
Available from: 2012-11-08 Created: 2012-11-07 Last updated: 2017-12-07Bibliographically approved

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