Direct analysis of cooperativity in multisubunit allosteric proteins

Abstract

Allosteric regulation of protein function is a fundamental phenomenon of major importance in many cellular processes. Such regulation is often achieved by ligand-induced conformational changes in multimeric proteins that may give rise to cooperativity in protein function. At the heart of allosteric mechanisms offered to account for such phenomenon, involving either concerted or sequential conformational transitions, lie changes in intersubunit interactions along the ligation pathway of the protein. However, structurefunction analysis of such homooligomeric proteins by means of mutagenesis, although it provides valuable indirect information regarding (allosteric) mechanisms of action, it does not define the contribution of individual subunits nor interactions thereof to cooperativity in protein function, because any point mutation introduced into homooligomeric proteins will be present in all subunits. Here, we present a general strategy for the direct analysis of cooperativity in multisubunit proteins that combines measurement of the effects on protein function of all possible combinations of mutated subunits with analysis of the hierarchy of intersubunit interactions, assessed by using high-order double-mutant cyclecoupling analysis. We show that the pattern of high-order intersubunit coupling can serve as a discriminative criterion for defining concerted versus sequential conformational transitions underlying protein function. This strategy was applied to the particular case of the voltage-activated potassium channel protein (Kv) to provide compelling evidence for a concerted all-or-none activation gate opening of the Kv channel pore domain. An direct and detailed analysis of the contribution of high-order intersubunit interactions to cooperativity in the function of an allosteric protein has not previously been presented. activation gate ͉ allosteric enzymes ͉ double-mutant cycles ͉ voltage-activated potassium channels ͉ non-additivity A llosteric regulation of protein function is often achieved by changes in protein conformation induced by the binding of substrate or other ligand molecules (1). In multisubunit proteins, such conformational changes may give rise to cooperativity in ligand binding. Several mechanistic models, in particular the Monod-Wymann-Changeux (MWC) model (2) and the Koshland-Némethy-Filmer (KNF) model (3), were developed in the 1960s to describe cooperativity in ligand binding. In both models, cooperativity is explained by binding-induced conformational changes in the subunits of the protein that may be concerted (MWC), sequential (KNF), or a combination of both (4). No matter the type of conformational change, cooperativity in ligand binding in all allosteric models is manifested by changes in intersubunit interactions along the ligation pathway of the protein (1).Despite the general acceptance of intersubunit interactions playing a fundamental role in determining the magnitude of cooperativity in ligand binding by an allosteric enzyme, structure-function analysis of such proteins throu...

Tetrameric assembly of KvLm K + channels with defined numbers of voltage sensors

“…In these studies, hetero-oligomers were assembled either by coexpression of mutant and WT subunits or by fixing the stoichiometry and arrangement of WT and mutant subunits in the oligomer through fusion of the genes encoding them in the desired order. Collectively, the inferences derived from findings with Shaker agree that the opening step is weakly voltage-dependent or voltage-independent (10,21,22), yet they differ on the degree of cooperativity underlying the transition (6,11,12).…”

“…In these studies, hetero-oligomers were assembled either by coexpression of mutant and WT subunits or by fixing the stoichiometry and arrangement of WT and mutant subunits in the oligomer through fusion of the genes encoding them in the desired order. Collectively, the inferences derived from findings with Shaker agree that the opening step is weakly voltage-dependent or voltage-independent (10, 21, 22), yet they differ on the degree of cooperativity underlying the transition (6,11,12).To explore the role of voltage sensors in the activation and opening of KvLm, we exploited the structural independence of the pore and sensor modules (26-30), together with the weak voltage-dependent gating of the sensorless pore module (PM) (S4-S5 linker-S5-S6) relative to the full-length channel (FL) (S1-S6) (31-33), to produce functional heterotetramers (Fig. 1B).…”

“…At the end of the activation pathway, each subunit is in an "activated-notopen" conformation (6,7) referring to the state of the sensor and pore, respectively. Although it is well established that each sensor moves independently during the early transitions in activation (8), the nature of the interactions between subunits in Kv channels underlying the transition from activated-not-open to open (the opening transition) remains unsettled (6,(9)(10)(11)(12). Structurally, the late kinetic transitions are considered to arise from conformational changes in the S4-S5 linker (7,13), whereas the final opening transition (6, 7) entails a change in conformation of S6, which forms the bundle-crossing of the pore (activation gate).…”

“…In Shaker, this has been achieved by blocking with 4-aminopyridine (7, 14, 15) or by mutations, including a single mutation at the beginning of the linker between the sensor and the pore (L382V) (3) or a triple mutation (ILT) in the S4 segment of the sensor (6,10,16,17). Alternatively, the assembly of hetero-oligomers composed of wild-type (WT) and mutant Kv subunits was explored as a means to examine inter-subunit interactions during voltage-dependent activation (4,6,(9)(10)(11)(12)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). In these studies, hetero-oligomers were assembled either by coexpression of mutant and WT subunits or by fixing the stoichiometry and arrangement of WT and mutant subunits in the oligomer through fusion of the genes encoding them in the desired order.…”

Concerted All-or-none Subunit Interactions Mediate Slow Deactivation of Human ether-à-go-go-related Gene K+ Channels

“…K V channel opening induced by membrane depolarization is mediated by a final, voltage-independent step that involves fully cooperative interactions between the S6 segments of all four subunits (37)(38)(39)(40). An outward splaying of the cytoplasmic ends of all four S6 segments widens this aperture (opens the pore) to permit K ϩ ion permeation.…”