{"id":2740,"date":"2015-10-21T16:10:00","date_gmt":"2015-10-21T15:10:00","guid":{"rendered":"http:\/\/www.blopig.com\/blog\/?p=2740"},"modified":"2015-10-21T16:26:21","modified_gmt":"2015-10-21T15:26:21","slug":"a-designed-conformational-shift-to-control-protein-binding-specificity","status":"publish","type":"post","link":"https:\/\/www.blopig.com\/blog\/2015\/10\/a-designed-conformational-shift-to-control-protein-binding-specificity\/","title":{"rendered":"A designed conformational shift to control protein binding specificity"},"content":{"rendered":"

\"header\"<\/a><\/p>\n

Proteins and their binding partners with complementary shapes form complexes. Fisher was onto something when he introduced the \u201ckey and lock mechanism\u201d in 1896. For the first time the shape of molecules was considered crucial for molecular recognition. Since then there have been various attempts to improve upon this theory in order incorporate the structural diversity of proteins and their binding partners.<\/p>\n

Koshland proposed the \u201cinduced fit\u201d mechanism in 1956, which states that interacting partners undergo local conformational changes upon complex formation to strengthen binding. An additional mechanism \u201cconformational selection\u201d was introduced by Monod, Wyman and Changeux who argued that the conformational change occurred before binding driven by the inherent conformational heterogeneity of proteins. Given a protein that fluctuates between two states A and B and a substrate C that only interacts with one of these states, the chance of complex formation depends on the probability of our protein being in state A or B. Furthermore, one could imagine a scenario where a protein has multiple binding partners, each binding to a different conformational state. This means that some proteins exists in an equilibrium of different structural states, which determines the prevalence of interactions with different binding partners.<\/p>\n

\"Figure1\"

Figure 1. The “pincer mode”.<\/p><\/div>\n

Based on this observation Michielssens et al. used various in-silico methods to manipulate the populations of binding-competent states of ubiquitin in order to change its protein binding behaviour. Ubiquitin is known to take on two equally visited states along the \u201cpincer mode\u201d (the collective motion describing the first PCA-eigenvector); closed and open.<\/p>\n


\n<\/span><\/p>\n

 <\/p>\n

 <\/p>\n

\"Figure2\"

Figure 2. A schematic of the conformational equilibrium of ubiquitin that can take on a closed or open state. Depending on its conformation i can bind different substrates.<\/p><\/div>\n

Different binding partners prefer either the closed, open or both states. By introducing point mutations in the core of ubiquitin, away from the binding interface, Michielssens et al. managed to shift the conformational equilibrium between open and closed states, thereby changing binding specificity.<\/p>\n

 <\/p>\n

 <\/p>\n

Point mutations were selected according to the following criteria:<\/p>\n

\u2043 residues must be located in the hydrophobic core
\n\u2043 binding interface must be unchanged by the mutation
\n\u2043 only hydrophobic residues may be introduced (as well as serine\/threonine)
\n\u2043 glycine and tryptophan were excluded because of their size<\/p>\n

\"Figure3\"<\/a>

Figure 3. Conformational preference of ubiquitin mutants. ddG_mut = dG_open – dG_closed.<\/p><\/div>\n

Fast growth thermal integration (FGTI), a method based on molecular dynamics, was used to calculate the relative de-\/stabilisation of the open\/closed state caused by each mutation. Interestingly, most mutations that caused stabilisation of the open states were concentrated on one residues, Isoleucine 36 (Slide 7).
\nFor the 15 most significant mutations a complete free energy profile was calculated using Umbrella sampling.<\/p>\n

\"Figure4\"

Figure 4. Free energy profiles for six different ubiquitin mutants, calculated using umbrella sampling simulations. Mutants preferring the closed substate are shown in red, open substate stabilizing mutants are depicted in blue, those without a preference are shown in gray.<\/p><\/div>\n

\"Figure5\"

Figure 5. Prediction of binding free energy differences between wild-type ubiquitin and different point mutations (ddG_binding = dG_binding,mutant\udbff\udc35 – dG_binding,wild-type).<\/p><\/div>\n

To further validate that they correctly categorised their mutants into stabilising the open or closed state, six X-ray structure of ubiquitin in complex with a binding partner that prefers either the open or closed state were simulated with each of their mutations. Figure 5\u00a0shows the change in binding free energy that is caused by the mutation in compatible, neutral and incompatible complexes (compatible may refer to an \u201copen favouring mutation\u201d (blue) in an open complex (blue) and vice versa).<\/p>\n

\"Figure6\"

Figure 6. Comparison of change in binding free energy predicted from the calculated results for ubiquitin and the experimental result.<\/p><\/div>\n

In their last step a selection of open and closed mutations was introduced into an open complex and the change in binding free energy was compared between experiment (NMR) and their simulations. For this example, their mutants behaved as expected and an increase in binding free energy was observed when the closed mutations were introduced into the open complex while only subtle changes were seen when the \u201ccompatible\u201d closed mutations were introduced.<\/p>\n

The authors suggest that in the future this computational protocol may be a corner stone to designing allosteric switches. However, given that this approach requires pre-existing knowledge and is tailored to proteins with well defined conformational states it may take some time until we discover its full potential.<\/p>\n","protected":false},"excerpt":{"rendered":"

Proteins and their binding partners with complementary shapes form complexes. Fisher was onto something when he introduced the \u201ckey and lock mechanism\u201d in 1896. For the first time the shape of molecules was considered crucial for molecular recognition. Since then there have been various attempts to improve upon this theory in order incorporate the structural […]<\/p>\n","protected":false},"author":29,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"nf_dc_page":"","wikipediapreview_detectlinks":true,"_monsterinsights_skip_tracking":false,"_monsterinsights_sitenote_active":false,"_monsterinsights_sitenote_note":"","_monsterinsights_sitenote_category":0,"ngg_post_thumbnail":0,"_jetpack_memberships_contains_paid_content":false,"footnotes":""},"categories":[1],"tags":[],"ppma_author":[519],"class_list":["post-2740","post","type-post","status-publish","format-standard","hentry","category-uncategorized"],"jetpack_featured_media_url":"","jetpack_sharing_enabled":true,"authors":[{"term_id":519,"user_id":29,"is_guest":0,"slug":"samuel","display_name":"Samuel Demharter","avatar_url":"https:\/\/secure.gravatar.com\/avatar\/9a7fc311ca91aec48d77ebd689fe2070f28c5442207cffd3adbd026c24de4d17?s=96&d=mm&r=g","0":null,"1":"","2":"","3":"","4":"","5":"","6":"","7":"","8":""}],"_links":{"self":[{"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/posts\/2740","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/users\/29"}],"replies":[{"embeddable":true,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/comments?post=2740"}],"version-history":[{"count":6,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/posts\/2740\/revisions"}],"predecessor-version":[{"id":2755,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/posts\/2740\/revisions\/2755"}],"wp:attachment":[{"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/media?parent=2740"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/categories?post=2740"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/tags?post=2740"},{"taxonomy":"author","embeddable":true,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/ppma_author?post=2740"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}